CN111848565A - Monoterpene bishydroxycoumarin compound, pharmaceutical composition, preparation method and application thereof - Google Patents
Monoterpene bishydroxycoumarin compound, pharmaceutical composition, preparation method and application thereof Download PDFInfo
- Publication number
- CN111848565A CN111848565A CN202010725360.3A CN202010725360A CN111848565A CN 111848565 A CN111848565 A CN 111848565A CN 202010725360 A CN202010725360 A CN 202010725360A CN 111848565 A CN111848565 A CN 111848565A
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- CN
- China
- Prior art keywords
- compound
- monoterpene
- bishydroxycoumarin
- racemate
- pharmaceutically acceptable
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- -1 Monoterpene bishydroxycoumarin compound Chemical class 0.000 title claims abstract description 36
- 229930003658 monoterpene Natural products 0.000 title claims abstract description 32
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- 125000004430 oxygen atom Chemical group O* 0.000 claims description 31
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
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Abstract
The invention relates to the field of natural pharmaceutical chemistry, and particularly provides a monoterpene bishydroxycoumarin compound, a pharmaceutical composition, a preparation method and an application thereof, wherein the monoterpene bishydroxycoumarin compound has a structure shown in a formula (I):
wherein R is1‑R6，X1‑X4As defined in the specification, the monoterpene dicoumarol compound provided by the invention can protect nerve cell damage by inhibiting the activity of acetylcholinesterase, and has the effects of anti-inflammation and antioxidation. Thus can be used forCan be used for preparing medicine for preventing and treating nervous system degenerative diseases such as senile dementia (also called Alzheimer disease), cholinergic nerve degenerative disease, learning and memory hypofunction, etc.
Description
Technical Field
The invention relates to the field of natural medicinal chemistry, and in particular relates to a monoterpene bishydroxycoumarin compound, a pharmaceutical composition, a preparation method and application thereof.
Background
Degenerative diseases of the central nervous system are a group of chronic and degenerative nervous system diseases, no measures for effectively controlling the disease process are clinically available for the diseases, and patients eventually lose the life ability and even die, for example, Alzheimer's disease, accounts for 60% -70% of dementia, and is one of the biggest threats to human health in the 21 st century. Recent statistics have shown that about 4000 million people over 60 years of age worldwide have AD, and in addition 770 million new cases per year, and have become an important public health problem worldwide. The etiology of AD has remained unclear to date. Therefore, the search for effective drugs for preventing and treating AD is urgent. Cholinergic nervous system dysfunction is one of the pathogenesis of AD, and regulation of cholinergic nervous system may become one of the ways to prevent and treat AD.
Trifoliate Evodia (Evodia lepta (Spreng.) Merr.) is a plant of Evodia of Rutaceae (Rutaceae), is mainly distributed in southern provinces and southeast Asia regions of China, and is a common Chinese herbal medicine in Lingnan. The Chinese medicine dictionary records that the tea has bitter taste and cold nature, has the effects of clearing heat and detoxicating, dispelling wind and removing dampness, is mainly used for treating sore throat, insect and snake bite, encephalitis, rheumatic osteodynia, eczema, dermatitis, sore and ulcer and the like, and is also one of important components of herbal tea in Guangdong areas.
At present, the research reports of chemical components of trigeminal bitter are more, and the chemical components mainly comprise dilute color, alkaloid, flavone and the like. Based on the previous researches, the research on the anti-senile dementia active lead compound is carried out on the stem of the trifurcate, and a monoterpene dicoumarol compound with obvious activity is found.
Disclosure of Invention
Therefore, one of the objects of the present invention is to provide monoterpene bishydroxycoumarin compounds, their enantiomers, diastereomers, cis-trans isomers, racemates, pharmaceutically acceptable salts or mixtures thereof.
The monoterpene bishydroxycoumarin compound has a structure shown in a formula (I),
wherein, with R1、R2、R3And R4Connected by
Independently selected from a single bond or a double bond;
the carbon atom with "#" and the carbon atom with "#" are chiral carbon atoms, the carbon atom with "#" is in R configuration, S configuration, or a combination thereof, and the carbon atom with "#" is in R configuration, S configuration, or a combination thereof;
X1、X2、X3and X4Independently selected from carbon atoms or oxygen atoms;
R1-R4independently selected from H, hydroxyl, oxygen atom, C1-C6Alkyl of (C)3-C6Cycloalkyl of, C1-C6Alkoxy group of (a);
R5、R6independently selected from H, hydroxy, C1-C5Alkyl of (C)3-C5Cycloalkyl of, C1-C5Alkoxy group of (2).
Further, R1、R2、R3、R4Independently selected from one of H, hydroxyl, oxygen atom, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, n-propoxy and isopropoxy;
when R is1、R2、R3Or R4When being an oxygen atom, bound to said oxygen atom
Is a double bond;
R5、R6independently selected from one of H, hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxy, ethoxy, n-propoxy and isopropoxy.
Further, X1And X2One of them is an oxygen atom and the other is a carbon atom; x3And X4One of them is an oxygen atomAnd one is a carbon atom;
R1and R2One of them is an oxygen atom and the other is a hydroxyl group; r3And R4One is an oxygen atom and the other is a hydroxyl group;
X1and R1Simultaneously oxygen atoms, or simultaneously not oxygen atoms; x2And R2Simultaneously oxygen atoms, or simultaneously not oxygen atoms; x3And R3Simultaneously oxygen atoms, or simultaneously not oxygen atoms; x4And R4Either simultaneously with oxygen atoms or simultaneously without oxygen atoms.
Further, the monoterpene bishydroxycoumarin compound has the following structures (II) to (V):
further, the monoterpene bishydroxycoumarin compound has a structure shown as follows:
the invention also aims to provide a preparation method of the monoterpene bishydroxycoumarin compound, which comprises the following steps:
1) extracting the trifoliate bitter stems with an organic solvent for one or more times to obtain an organic solvent extract;
2) dispersing the organic solvent extract with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and recovering ethyl acetate extract;
3) separating the ethyl acetate extract by a silica gel chromatographic column, carrying out gradient elution by using cyclohexane/ethyl acetate as an eluent to obtain a crude component, and carrying out one-time MCI column chromatography, one-time reverse ODS column chromatography and one-time Sephadex LH-20 column chromatography on the obtained crude component to obtain a column chromatography component of the monoterpene bishydroxycoumarin compound;
4) separating and refining column chromatography component containing monoterpene bishydroxycoumarin compound by HPLC to obtain the final product.
Further, the production method satisfies at least one of the following items (1) to (8);
(1) in the step 1), the organic solvent is at least one selected from n-hexane, dichloromethane, ethyl acetate, n-butanol, methanol, 50-95% methanol aqueous solution, ethanol and 50-95% ethanol aqueous solution;
(2) in the step 1), the extraction is reflux extraction or ultrasonic extraction, preferably reflux extraction, wherein the time of reflux extraction is 1-3h, and the times are 2-5;
(3) in the step 1), adding an organic solvent with 8-12 times of volume;
(4) in the step 1), the organic solvent extract is preferably an extract obtained by extracting trifurcate bitter stems and then evaporating to dryness;
(5) in the step 3), in the separation process of one MCI column chromatography, the column chromatography adopts methanol and water with the volume ratio of 2: 3 → 1: 0 to carry out gradient elution; performing gradient elution by adopting methanol and water with the volume ratio of 3: 2 → 1: 0 for the first reverse ODS column chromatography;
(6) in the step 3), in the separation process of the silica gel chromatographic column, cyclohexane and ethyl acetate with the volume ratio of 100: 1 → 0: 1 are used as eluent for gradient elution;
(7) in the step 3), methanol or ethanol is adopted as an eluent for elution in the Sephadex LH-20 column chromatography separation process;
(8) in the step 4), acetonitrile or methanol water solution with the volume percentage of 50-75% is adopted as a mobile phase in the separation and refining process; after separation and refining, the solution is resolved by a chiral HPLC column.
The invention also provides a pharmaceutical composition which comprises any one of the monoterpene bishydroxycoumarin compounds, enantiomers, diastereomers, cis-trans-isomers, racemates, pharmaceutically acceptable salts or mixtures thereof, and optionally a pharmaceutically acceptable carrier.
Further, the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integration agents, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, polymeric matrix materials, and film-forming materials, based on the total mass of the pharmaceutical composition;
the pharmaceutical composition is gel, cream, tablet, capsule, powder, mixture, pill, granule, solution, syrup, soft extract, suppository, aerosol, emplastrum, ointment, injection, spray, liniment, tincture, wet dressing, paste, lotion or sustained and controlled release preparation.
The monoterpene dicoumarol compound, enantiomer, diastereomer, cis-trans isomer, raceme, pharmaceutically acceptable salt or mixture thereof, or the application of the pharmaceutical composition in preparing acetylcholinesterase inhibitor.
The monoterpene bishydroxycoumarin compound, the enantiomer, the diastereomer, the cis-trans isomer, the racemate, the pharmaceutically acceptable salt or the mixture thereof, or the application of the pharmaceutical composition in preparing the drugs for preventing or treating neurodegenerative diseases.
Further, the neurodegenerative disease is senile dementia, hypomnesis, brain tissue degenerative disease syndrome or cholinergic neurodegenerative disease.
The technical scheme of the invention has the following advantages:
the monoterpene bishydroxycoumarin compound provided by the invention has the effects of remarkably improving scopolamine-induced nerve cell injury, remarkably inhibiting the activity of acetylcholinesterase, resisting inflammation and oxidation, and can be used for preventing and treating senile dementia (also called Alzheimer disease), cholinergic neurodegenerative diseases, learning and memory impairment and other nervous system degenerative diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the results of oxidative stress test in Experimental example 1 of the present invention, wherein A is MDA level and B is SOD activity level;
FIG. 2 is a graph showing the results of the neuroinflammation test in Experimental example 1 of the present invention, wherein A is IL-1. beta. and B is the IL-6 level;
FIG. 3 is a high resolution mass spectrometry (HR-ESI-MS) pattern of racemate 1 in example 1 of the present invention;
FIG. 4 shows a scheme for preparing racemate 1 in example 1 of the present invention1H-NMR spectrum (400 MHz);
FIG. 5 shows a scheme for preparing racemate 1 in example 1 of the present invention13C-NMR spectrum (100 MHz);
FIG. 6 is a two-dimensional nuclear magnetic resonance (COSY NMR) spectrum of racemate 1 in example 1 of the present invention;
FIG. 7 is a heteronuclear single quantum coherent nuclear magnetic resonance (HSQC NMR) spectrum of racemate 1 in example 1 of the present invention;
FIG. 8 shows a scheme for preparing racemate 1 in example 1 of the present invention1Heteronuclear multiple carbon correlation nuclear magnetic resonance (HMBC NMR) spectrum of H;
FIG. 9 is a NOE (ROESY NMR) spectrum of a rotating coordinate system of racemic modification 1 in example 1 of the present invention;
FIG. 10 is a gas phase ECD detection spectrum of Compound 1 in example 1 of the present invention.
FIG. 11 is a gas phase ECD detection profile of Compound 2 of example 1 of the present invention.
FIG. 12 is a high resolution mass spectrometry (HR-ESI-MS) pattern of racemate 2 in example 1 of the present invention;
FIG. 13 shows racemate 2 in example 1 of the present invention1H-NMR spectrum (400 MHz);
FIG. 14 shows racemate 2 in example 1 of the present invention13C-NMR spectrum (100 MHz);
FIG. 15 is a two-dimensional nuclear magnetic resonance (COSY NMR) spectrum of racemate 2 in example 1 of the present invention;
FIG. 16 is a heteronuclear single quantum coherent nuclear magnetic resonance (HSQC NMR) spectrum of racemate 2 in example 1 of the present invention;
FIG. 17 shows racemate 2 in example 1 of the present invention1Heteronuclear multiple carbon correlation nuclear magnetic resonance (HMBC NMR) spectrum of H;
FIG. 18 is a NOE (ROESY NMR) spectrum of a rotating coordinate system of racemic modification 2 in example 1 of the present invention;
FIG. 19 is a high resolution mass spectrometry (HR-ESI-MS) pattern of racemate 3 in example 1 of the present invention;
FIG. 20 shows racemate 3 in example 1 of the present invention1H-NMR spectrum (400 MHz);
FIG. 21 shows racemate 3 in example 1 of the present invention13C-NMR spectrum (100 MHz);
FIG. 22 is a two-dimensional nuclear magnetic resonance (COSY NMR) spectrum of racemic modification 3 in example 1 of the present invention;
FIG. 23 is a heteronuclear single quantum coherent nuclear magnetic resonance (HSQC NMR) spectrum of racemate 3 in example 1 of the present invention;
FIG. 24 shows racemate 3 in example 1 of the present invention1Heteronuclear multiple carbon correlation nuclear magnetic resonance (HMBC NMR) spectrum of H;
FIG. 25 is a NOE (ROESY NMR) spectrum of racemic modification 3 in example 1 of the present invention;
FIG. 26 is a high resolution mass spectrometry (HR-ESI-MS) pattern of racemate 4 in example 1 of the present invention;
FIG. 27 shows racemate 4 in example 1 of the present invention1H-NMR spectrum (400 MHz);
FIG. 28 shows a scheme for preparing racemate 4 in example 1 of the present invention13C-NMR spectrum (100 MHz);
FIG. 29 is a two-dimensional nuclear magnetic resonance (COSY NMR) spectrum of racemic modification 4 in example 1 of the present invention;
FIG. 30 is a heteronuclear single quantum coherent nuclear magnetic resonance (HSQC NMR) spectrum of racemate 4 in example 1 of the present invention;
FIG. 31 shows a scheme for the production of racemate 4 in example 1 of the present invention1Heteronuclear multiple carbon correlation nuclear magnetic resonance (HMBC NMR) spectrum of H;
FIG. 32 is a NOE (ROESY NMR) spectrum of racemic modification 4 in example 1 of the present invention;
FIG. 33 is a high resolution mass spectrometry (HR-ESI-MS) spectrum of racemate 5 in example 1 of the present invention;
FIG. 34 shows a scheme for preparing racemate 5 in example 1 of the present invention1H-NMR spectrum (400 MHz);
FIG. 35 shows a scheme for preparing racemate 5 in example 1 of the present invention13C-NMR spectrum (100 MHz);
FIG. 36 is a two-dimensional nuclear magnetic resonance (COSY NMR) spectrum of racemic body 5 in example 1 of the present invention;
FIG. 37 is a heteronuclear single quantum coherent nuclear magnetic resonance (HSQC NMR) spectrum of racemate 5 in example 1 of the present invention;
FIG. 38 shows a scheme for preparing racemate 5 in example 1 of the present invention1Heteronuclear multiple carbon correlation nuclear magnetic resonance (HMBC NMR) spectrum of H;
FIG. 39 is a NOE (ROESY NMR) spectrum of racemic modification 5 in example 1 of the present invention.
Detailed Description
The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially. The following experiments were carried out using trifurcate-Evodia lepta (Spreng.) Merr. stems from Mappia city, Guangdong province.
Experimental example 1
First, experiment purpose
The anti-senile dementia effect of the compounds 1-10 is investigated by adopting a SH-SY5Y cell model induced by scopolamine as a molding agent and researching the neuroprotective effect of the compounds, the influence of oxidative stress and inflammation.
Second, Experimental methods
1. Experimental Material
Preparing a solution of a sample to be detected: the test samples were pure compounds 1 to 10 isolated and purified in example 1, and each of the compounds 1 to 10 was precisely weighed and dissolved in DMSO to prepare a mother solution containing each of the compounds 1 to 10 at a concentration of 30mM, in which mM is mmol/L, and the mother solutions of each compound were diluted with DMSO to prepare solutions having concentration gradients of 0.1nM, 1nM, 10nM and 100nM, in which nM is nmol/L.
Preparation of positive control solution (donepezil solution): donepezil was precisely weighed and dissolved in DMSO to prepare a stock solution containing 30mM of donepezil, and the stock solution was diluted with DMSO to obtain solutions having the following concentration gradients of 0.1nM, 1nM, 10nM, and 100nM, respectively.
2. Cell lines and subculture of cells
Human neuroblastoma SH-SY5Y cells were purchased from ATCC cell bank. And (3) recovering the cells: taking out the freezing tube from the liquid nitrogen, immediately placing into a 37 ℃ water bath kettle, rapidly shaking until the freezing liquid is completely dissolved, and completing rewarming within 5 min; transferring a centrifugal tube into a super clean bench, adding 5ml of DMEM culture solution, and uniformly mixing; 400g, centrifuging for 5min, and discarding the supernatant; adding new DMEM complete culture solution into the cell sediment, gently mixing, transferring into a culture bottle, adding sufficient DMEM culture solution, standing at 37 deg.C, and adding 5% CO2Cultured in an incubator.
Passage of cells: DMEM culture medium containing 10% inactivated FBS at 37 deg.C and 5% CO2SH-SY5Y cells were cultured in a cell culture incubator.
3. Vitality testing method
Sample (I)The protection effect on scopolamine-induced cell injury is determined by MTT method, and SH-SY5Y cells in logarithmic phase are resuspended at 5 × 103One of the cells was inoculated into a 96-well plate and cultured for 24 hours, 200. mu.L per well, and was randomly divided into a compound 1-10 group, a positive control group, a blank control group, and a scopolamine model group. Adding 20 μ L of the prepared test sample solution containing compounds 1-10 at different concentrations into each well of compound 1-10 group, adding 20 μ L of donepezil solution at different concentrations into each well of positive control group, adding 20 μ L of DMSO into each well of blank control group and scopolamine model group, culturing at 37 deg.C for 24 hr, adding 20 μ L of scopolamine aqueous solution containing scopolamine 2mmol/L into each well of compound 1-10 group, scopolamine model group and positive control group, and culturing at 37 deg.C for 5% CO2The culture was continued in the carbon dioxide incubator for 24 hours, MTT 14. mu.L was added to each well, and the incubation was continued for 3 hours, after which the culture medium was removed, and 150. mu.L DMSO was added to each well and the mixture was shaken on a shaker for 10 minutes to dissolve the crystals sufficiently. And oscillating and mixing the mixture on an oscillator to fully dissolve the reduction product. Selecting 570nm wavelength, measuring the light absorption value (A value) of each well on an enzyme-linked immunosorbent assay, setting six parallel wells for the sample and the blank control group in the experiment, taking the average value of the A value, and obtaining the cell inhibition rate (%) according to the formula [1- (compound 1-10A value/blank control group A value)]X 100% cell inhibition and then EC50. Each experiment was repeated three times.
4. Determination of acetylcholinesterase Activity: preparing a sample solution of compounds 1-10 with the concentration of 0.1nM, 1nM, 10nM and 100nM according to the above step 1, treating SH-SY5Y cells according to the methods of steps 2 and 3, collecting cells of the blank control group, the scopolamine model group, the positive control group and the compounds 1-10 group, adding cells before MTT and cell culture solution in the above step 3, centrifuging, taking cell culture solution supernatant, and testing the activity of acetylcholinesterase in the cell culture solution supernatant of each group by using an AchE kit (Nanjing institute of bioengineering) according to the method in the kit instruction.
5. Determination of oxidative stress, determination of Interleukin 1 β (IL-1 β) and Interleukin-6 (IL-6): according to the results of the cell viability assay, refer to "measured in step 1Preparation of sample solutions "preparation of EC for Compounds 1-10, respectively50Concentration of sample solution and EC50Donepezil solutions at concentrations, specifically, mother liquors at 30mM concentrations of each compound were diluted with DMSO to sample solutions at concentrations of 20.9nM (compound 1), 18.6nM (compound 2), 10.7nM (compound 3), 13.1nM (compound 4), 15.4nM (compound 5), 15.6nM (compound 6), 17.7nM (compound 7), 17.1nM (compound 8), 16.3nM (compound 9), and 15.9nM (compound 10), respectively; stock solutions with a donepezil concentration of 30mM were diluted to 16.9nM in DMSO. SH-SY5Y cells were cultured according to the method of step 2, and SH-SY5Y cells at logarithmic phase were resuspended at 5X 103One of the cells was inoculated into a 96-well plate and cultured for 24 hours, 200. mu.L per well, and was randomly divided into a compound 1-10 group, a positive control group, a blank control group, and a scopolamine model group. Compounds 1-10 groups EC formulated as described above were added to each well5020 mu L of sample solution with concentration, 20 mu L of donepezil solution with concentration of 16.9nM prepared in each hole of the positive control group, 20 mu L of DMSO respectively in each hole of the blank control group and the scopolamine model group, continuing to culture at 37 ℃ for 24h, 20 mu L of scopolamine aqueous solution with concentration of 2mmol/L in each hole of the cells of the compound 1-10 groups, the scopolamine model group and the positive control group, and culturing at 37 ℃ at 5% CO2The culture is continued for 24 hours in the carbon dioxide incubator, cells and cell culture solution are collected and centrifuged, and cell culture solution supernatant is taken.
Respectively taking cell culture solution supernatants, and testing the content of Malondialdehyde (MDA) in the cell culture solution supernatants of each group by using an MDA kit (Nanjing institute of bioengineering) according to the method recorded in the kit specification; testing the activity of superoxide dismutase (SOD) in the supernatant of each group of cell culture solution by using an SOD enzyme activity kit (Nanjing institute of bioengineering) according to the method recorded in the kit specification; the supernatant of each cell culture was assayed for IL-1 β, IL-6 content using the IL-1 β, IL-6ELISA kit (Thermo Fisher scientific) by ELASA assay according to the protocol described in the kit instructions.
6. Statistical analysis: the detection results are expressed by mean value + -SEM, statistical data analysis is carried out on the group of samples to be detected and the group of blank control groups, and P <0.05 represents that the significant difference exists.
Second, experimental results
1. Cell viability test results
TABLE 1 cell viability test results Table
1EC50: half maximal effect concentration.
The results are shown in Table 1, and show that the compound 1-10 can protect scopolamine-induced cell injury, and the EC thereof50The minimum concentration was 13.1. + -. 0.7. mu.M. The compound is shown to have neuroprotective effect.
2. Results of acetylcholinesterase Activity test
The results are shown in Table 2, and the above experimental results show that the compounds 1-10 can inhibit the significant increase of the acetylcholinesterase activity induced by scopolamine and the IC of the compound is IC, compared with the scopolamine model group50The minimum concentration is 7.3 +/-0.7 nM, which shows that the compound has the effect of inhibiting the activity of acetylcholinesterase.
TABLE 2 results of acetylcholinesterase Activity
1IC50: half inhibitory concentration.
3. Results of oxidative stress test
The results are shown in fig. 1, and the formulas 1-10 correspond to the compounds 1-10, and the experimental results show that the compounds 1-10 can obviously relieve the increase degree of scopolamine-induced MDA level and can improve the decrease degree of scopolamine-induced antioxidase SOD activity compared with a scopolamine model group. The compound is shown to have the function of improving the antioxidant capacity.
4. Neuroinflammation test results
The results are shown in FIG. 2, in which formula 1-formula 10 corresponds to compound 1-10, and the experimental results show that compound 1-10 can significantly alleviate the increase of the levels of IL-1 β and IL-6 induced by scopolamine, compared with the scopolamine model group. The compound is shown to have anti-inflammatory effect.
In fig. 1-2, a indicates significant difference compared to the blank control group, p <0.01, #indicatessignificant difference compared to the scopolamine model group, p < 0.01.
In conclusion, the compound 1-10 provided by the invention has protective effect on nerve cells of a scopolamine-induced senile dementia model, can inhibit acetylcholinesterase activity, and has anti-inflammatory and antioxidant effects.
EXAMPLE 1 preparation of Compounds 1-10
(1) Reflux-extracting herba Ixeritis Sonchifoliae with 70% ethanol water solution 12 times the weight of herba Ixeritis Sonchifoliae at 92 deg.C for 2 hr, and collecting ethanol extractive solution; adding 70% ethanol water 10 times the weight of the trifoliate root into the residue, reflux-extracting once, heat-refluxing at 92 deg.C for 2 hr, and collecting ethanol extractive solution; adding 70% ethanol water solution 8 times the weight of the trifoliate root into the residue, reflux-extracting for one time, heat refluxing for 2 hr, and collecting ethanol extractive solution; mixing the ethanol extractive solutions, and concentrating under reduced pressure until no ethanol smell is detected to obtain 4kg ethanol extract.
(2) Dissolving 4kg of ethanol extract with distilled water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and recovering ethyl acetate extract (namely the extract of the invention).
(3) The ethyl acetate extract was chromatographed on silica gel with six gradients of cyclohexane/ethyl acetate, 100: 1, 5: 1, 10: 1, 20: 1, 50: 1, 0: 1 by volume, receiving 10 fractions per 500ml, designated Fr-1 to Fr-10 respectively. Performing MCI column chromatography on Fr-4 (flow parts of cyclohexane/ethyl acetate with the volume ratio of 5: 1), eluting with four gradients of methanol/water with the volume ratios of 2: 3, 1: 1, 3: 2 and 1: 0, receiving one flow part per 500ml, and receiving 7 flow parts which are respectively marked as Fr-4-1 to Fr-4-7. Performing reverse ODS column chromatography on Fr-4-5 (flow parts of methanol/water with the volume ratio of 1: 1), eluting with four gradients of methanol/water with the volume ratios of 3: 2, 7: 3, 8: 2 and 0: 1, receiving one flow part per 400ml, and receiving 8 flow parts which are respectively marked as Fr-4-5-1 to Fr-4-5-8. And (3) carrying out Sephadex LH-20 column chromatography separation on the flow Fr-4-5-5 (flow with the volume ratio of methanol to water of 8: 2) for one time, eluting with methanol as an eluent, receiving one flow per 500ml, and receiving 5 flows which are respectively marked as Fr-4-5-5-1 to Fr-4-5-5-5.
(4) Fr-4-5-5-4 is separated and refined by HPLC, acetonitrile water solution with the volume fraction of 70 percent is used as a mobile phase, the flow rate is 7.0ml/min, the detection wavelength is 210nm, racemes 1 to 5 are obtained, and the racemes 1 to 5 are racemes through optical rotation determination.
(5) Taking the racemate 1, and resolving by using a chiral HPLC column, wherein 70 volume parts of acetonitrile aqueous solution is used as a mobile phase, the flow rate is 7.0ml/min, and the detection wavelength is 210nm to obtain a compound 1(12mg) and a compound 2(17 mg); taking the racemate 2, and resolving by using a chiral HPLC column, wherein 70 volume parts of acetonitrile aqueous solution is used as a mobile phase, the flow rate is 7.0ml/min, and the detection wavelength is 210nm, so that a compound 3(14mg) and a compound 4(28mg) are obtained; taking the racemate 3, and resolving by using a chiral HPLC column, wherein 90 volume parts of acetonitrile aqueous solution is used as a mobile phase, the flow rate is 7.0ml/min, and the detection wavelength is 210nm, so that a compound 5(39mg) and a compound 6(15mg) are obtained; taking the racemate 4, and resolving by using a chiral HPLC column, wherein 90 volume parts of acetonitrile aqueous solution is used as a mobile phase, the flow rate is 7.0ml/min, and the detection wavelength is 210nm, so that a compound 7(18mg) and a compound 8(19mg) are obtained; taking the racemate 5, and carrying out resolution by using a chiral HPLC column, taking 70 volume parts of acetonitrile aqueous solution as a mobile phase, wherein the flow rate is 7.0ml/min, and the detection wavelength is 210nm, so as to obtain a compound 9(26mg) and a compound 10(33 mg).
Example 2 characterization of racemates 1-5 and Compounds 1-10
The racemate 1 is white amorphous powder, HR-ESI-MThe S map gives the excimer ion peak M/z 457.1649[ M + H ]]+(calcd. for 457.1651), bonding13The C NMR spectrum of the compound 1 and the compound 2 shows that the molecular formulas are C28H24O6. Ultraviolet (UV) spectrum shows characteristic absorption (lambda) of coumarinmax202nm,246nm,333 nm); infrared Spectrum (IR) showed hydroxyl absorption (3647 cm)-1) Carbonyl absorption (1716 cm)-1)。
1In the H-NMR spectrum, characteristic hydrogen signals of a group of coumarin parent nuclei are observed in a low field region:H6.10(1H, d, J ═ 9.4Hz, H-3) andH7.79(1H, d, J ═ 9.4Hz, H-4); a set of double bond proton signals of E configuration:H5.96(1H, d, J ═ 16.4Hz, H-9') andH6.71(1H, d, J ═ 16.4Hz, H-10'); in addition, two olefinic hydrogen proton signals are present in the low field region:H5.30(1H, brs, H-10) andH7.56(1H, s, H-4'); and a group of benzene rings are proton signals of ABX system:H7.38(1H, d, J ═ 8.5Hz, H-5 '), 6.75(1H, dd, J ═ 8.5,2.3Hz, H-6 ') and 6.64(1H, d, J ═ 2.3Hz, H-8 ').13The C-NMR spectrum showed 28 carbon signals including 2 methyl groups, 2 methylene groups, 12 methine groups, 14 quaternary carbons, and further analysis of the HSQC spectral data suggested the possible presence of two coumarin units.
In that1H-1In the H COSY NMR spectrum, there are five sets of proton coupling systems: h-3 and H-4, H-5 'and H-6, H-9 and H-10, H-5' and H-6 ', H-12 and H-12', combine multiple relevant signals in HMBC: h-3, H-8 and C-4a, H-4 and C-2, C-8a, H-8 and C-6, and determining one structural Unit of a coumarin Unit (Unit A) in the compound; according to the relevant signals in the other HMBC: h-3 ', H-8' and C-4a ', H-4' and C-2 ', C-8 a', H-8 'and C-6', and also defines another coumarin Unit (Unit B) of the compound. According to three groups of proton coupling systems: h-9 and H-10, H-9 ' and H-10 ', H-12 and H-12 '; and four sets of correlated signals in the HMBC spectrum: h-9 and C-11, H3-13 and C-10, C-12, H-9 'and C-11', H-10 'and C-12', C-13 'indicating that the compound may have a six-membered ring with a methyl group attached to C-11' and C-11 ', respectively, and a vinyl group attached to C-11', thereby definingAnd another structural Unit (Unit C). Finally, by two sets of correlated signals in the HMBC spectrum: h-5 and C-9, H-9 ' and C-11 ' and C-4 ' finally determine the connection mode of the three structural units, namely Unit A and Unit C are connected with one carbon single bond formed by C-6 and C-9, and Unit B and Unit C are connected with one carbon single bond formed by C-3 ' and C-9 '.
A set of relevant signals was observed in the ROESY NMR spectra: h-9 and H313 ', determining the relative configuration of compound 1 and compound 2 (9R, 11' R or 9S,11' S), finally chiral resolution of racemate 1 by means of the chiral column of step (5) of example 1, obtaining a pair of enantiomers, and determining the absolute configuration by comparing the experimental and calculated values of their ECDs (figures 10 and 11): compound 1 is 9R,11' R [ (+) -Compound 1]The compound 2 is 9S,11' S [ (-) -compound 2]They are named (+) -Dievodiletin C, (-) -Dievodiletin C, respectively.
TABLE 3 racemic modification 1-31H NMR and13c NMR data (CD)3OD，J＝Hz)
TABLE 4 racemic modification 4-51H NMR and13c NMR data (CD)3OD,J＝Hz)
The racemate 2 is white amorphous powder, and the HR-ESI-MS shows an excimer peak M/z 457.1648[ M + H ]]+(calcd. for 457.1651), bonding13C NMR spectrum of the formula C28H24O6Suggest that it is a combination of Compound 1 and2 are each an isomer. Ultraviolet (UV) spectrum shows characteristic absorption (lambda) of coumarinmax244nm and 328 nm); infrared Spectrum (IR) showed hydroxyl absorption (3647 cm)-1) Carbonyl absorption (1716 cm)-1)。
1In the H-NMR spectrum, two signals of olefinic hydrogen protons are observed in the low field region:H5.29(1H, brs, H-10) andH7.69(1H, s, H-4'); and two groups of benzene rings are proton signals of an ABX system:H7.42(1H, d, J ═ 8.5Hz, H-5 '), 6.76(1H, dd, J ═ 8.5,2.3Hz, H-6 '), 6.65(1H, d, J ═ 2.2Hz, H-8 '), andH7.38(1H,d,J＝8.5Hz,H-5)、6.71(1H,dd,J＝8.5,2.3Hz,H-6)、6.60(1H,d,J＝2.3Hz,H-8)。13the C-NMR spectrum showed that this compound had 28 carbons, 2 methyl groups, 2 methylene groups, 12 methine groups, and 14 quaternary carbons.
In that1H-1In the H COSY NMR spectrum, there are 5 groups of proton coupling systems: h-5 and H-6, H-5 ' and H-6 ', H-9 and H-10, H-9 ' and H-10 ', H-12 and H-12 '; and determining two structural units of Unit A and Unit B by combining HMBC spectrums. According to four sets of correlation signals in HMBC: h-9 and C-11, H3-13 and C-10, C-12, H-9 ' and C-11 ', H-10 ' and C-12 ', C-13 ' define the UnitC building block; the above NMR information further proves that racemate 2 and racemate 1 are isomers each other, and both contain three structural units of Unit a, Unit B and Unit C, but the connection modes are different, and finally, according to two groups of related signals in HMBC: h-4 and C-9, H-9 ' and C-4 ' with C-2 ', finally defining the connection mode of three structural units.
Also, in the ROESY NMR spectrum, there is a set of relevant signals: h-9 and H3-13 ', i.e. the relative configuration of compound 3 and compound 4, as in compound 1 and compound 2 (9R, 11' R or 9S,11' S), are designated (+) -divaricatin D, (-) -divaricatin D, respectively.
The racemate 3 is white amorphous powder, and the HR-ESI-MS shows an excimer peak M/z 457.1642[ M + H ]]+(calcd. for 457.1642), bonding13C NMR spectrum of the formula C28H24O6This suggests that they are isomers with compounds 1 to 4. Ultraviolet (UV) spectrum shows characteristic absorption (lambda) of coumarinmax201nm,255nm,329 nm); infrared Spectrum (IR) showed hydroxyl absorption (3647 cm)-1) And carbonyl absorption (1716 cm)-1)。
1In an H-NMR spectrum, a low field region has characteristic proton signals of a group of coumarin mother rings:H6.18(1H, d, J ═ 9.4Hz, H-3') andH7.89(1H, d, J ═ 9.4Hz, H-4'); two olefinic hydrogen proton signals:H5.31(1H, d, H-10) andH7.55(1H, s, H-4); proton signal of a group of benzene ring ABX systems:H7.44(1H,d,J＝8.5Hz,H-5)、6.77(1H,dd,J＝8.5,2.3Hz,H-6)、6.67(1H,d,J＝2.3Hz,H-8)。13the C-NMR spectrum showed that the compound also had 28 carbons, 2 methyl groups, 2 methylene groups, 12 methine groups, and 14 quaternary carbons.
In that1H-1In the H COSY spectrum, there are 5 groups of proton coupling systems: h-5 and H-6, H-3 ' and H-4 ', H-9 and H-10, H-9 ' and H-10 ', H-12 and H-12 '; and determining two structural units of Unit A and Unit B by combining HMBC spectrums. According to four sets of correlation signals in HMBC: h-9 and C-11, H3-13 and C-10, C-12, H-9 ' and C-11 ', H-10 ' and C-12 ', C-13 ' define the UnitC building block; the NMR information further proves that the racemic modification and the racemate 1 are isomers, contain Unit A, Unit B and Unit C, have differences in connection modes, and finally, according to two groups of related signals in HMBC: h-4 and C-9, H-9 ' and C-2 ', C-4 ' finally define the connection mode of the three structural units.
A set of relevant signals (H-9 and H) in the ROESY NMR spectra3-13 '), also suggesting that the relative configuration of compound 5 and compound 6 is the same as that of compound 1 and compound 2 (9R, 11' R or 9S,11' S), which are designated as (+) -divaricatin E, (-) -divaricatin E, respectively.
The racemate 4 is white amorphous powder, and the HR-ESI-MS shows an excimer peak M/z 457.1642[ M + H ]]+(calcd.for 457.1651)，13C NMR spectrum of the formula C28H24O6It is suggested that it is an isomer with each of the compounds 1 to 6. Ultraviolet (UV) spectrum shows characteristic absorption (lambda) of coumarinmax201nm,299nm,327 nm); infrared Spectrum (IR) showed hydroxyl absorption (3647 cm)-1) And carbonyl absorption (1716 cm)-1)。
1In the H-NMR spectrum, a low field region shows that a group of characteristic proton signals of coumarin mother rings exist:H6.11(1H, d, J ═ 9.4Hz, H-3 ') and 7.58(1H, d, J ═ 9.4Hz, H-4'); one set of olefinic hydrogen proton signals:H6.26(1H, d, J-12.8 Hz, H-9 ') and 5.76(1H, d, J-12.8 Hz, H-10'); two olefinic hydrogen proton signals:H5.13(1H, brs, H-10) and 7.43(1H, s, H-4); and a group of benzene rings are proton signals of ABX system:H7.39(1H, d, J ═ 8.5Hz, H-5), 6.76(1H, dd, J ═ 8.5,2.3Hz, H-6) and 6.72(1H, d, J ═ 2.3Hz, H-8).13The C-NMR spectrum showed that this compound had 28 carbons, 2 methyl groups, 2 methylene groups, 12 methine groups, and 14 quaternary carbons.
In that1H-1In the H COSY spectrum, 5 groups of proton coupling systems were observed: h-5 and H-6, H-3 ' and H-4 ', H-9 and H-10, H-9 ' and H-10 ', H-12 and H-12 ', and HMBC spectra are combined to determine two structural units of Unit A and Unit B. According to three sets of correlation signals in HMBC: h-9 and C-11, H 313 and C-10, C-12, H-10 'and C-12', C-9, defining the Unit C building block. From the NMR information, compound 7 and compound 8 are also composed of three units, Unit a, Unit B and Unit C, and finally, two groups of related signals in HMBC are shown: h-4 and C-9, H-9 'and C-5', determined that the three building blocks were linked in the same manner as racemate 3, the only difference being the coupling constant (J ═ 12.8Hz) of H-9 'and H-10' for racemate 4, suggesting that this position is of Z configuration, and therefore they are a pair of coumarin dimers that are Z/E isomers (or cis-trans isomers) of each other.
A set of correlated signals (H-9 and H) in the ROESY NMR spectra3-13') are also suggestedThe relative configuration of compound 7 and compound 8 is the same as that of compound 1 and compound 2, namely 9R,11'R or 9S,11' S, which are named as (+) -Dievodiletin F, (-) -Dievodiletin F, respectively.
The racemate 5 is white amorphous powder, and the HR-ESI-MS spectrum shows an excimer ion peak M/z 457.1647[ M + H ]]+(calcd. for 457.1651), bonding13C NMR spectrum of the formula C28H24O6Compound 9 is suggested to be an isomer of compound 10 and compounds 1-8. Ultraviolet (UV) spectrum shows characteristic absorption (lambda) of coumarinmax203nm,300nm,329 nm); infrared Spectrum (IR) showed hydroxyl absorption (3647 cm)-1) And carbonyl absorption (1716 cm)-1)。
1In the H NMR spectrum, characteristic proton signals of two groups of coumarin mother rings are observed in a low field:H6.20(1H, d, J ═ 9.4Hz, H-3), 7.88(1H, d, J ═ 9.4Hz, H-4), andH6.13(1H, d, J ═ 9.4Hz, H-3), 7.46(1H, d, J ═ 9.4Hz, H-4); one set of olefinic hydrogen proton signals:H6.04(1H, d, J ═ 12.8Hz, H-9) and 5.78(1H, d, J ═ 12.8Hz, H-10), the coupling constants of H-9 and H-10 (J ═ 12.8Hz) suggest that racemate 5 is in the Z configuration with racemate 4.13The C-NMR spectrum showed that this compound had 28 carbons, 2 methyl groups, 2 methylene groups, 12 methine groups, and 14 quaternary carbons.
In that1H-1In the H COSY spectrum, there are 5 groups of proton coupling systems: h-3 and H-4, H-9 and H-10, H-12 and H-12; and determining two structural units of Unit A and Unit B by combining HMBC spectrums. According to three sets of correlation signals in HMBC: h-9 and C-11, H 313 and C-10, C-12, H-10 and C-12, C-9, thus determining the C structural Unit, and the NMR information further proves that the racemate 5 and the racemate 1 are isomers with each other and all contain three structural units of Unit A, Unit B and Unit C, and finally, according to two groups of related signals in HMBC: h-5 and C-9, H-9 and C-5, finally define the connection mode of the three structural units.
A set of related signals (H-9 and H) present in the ROESY NMR spectra3-13), suggesting that the relative configuration of compound 9 and compound 10 is the same as that of compound 1 and compound 2, and is also 9R,11'R or 9S,11' S, which are designated as (+) -divaricatin F, (-) -divaricatin F, respectively.
Wherein, racemic modifications 1 to 5131 or 2 data on the C-NMR spectrum are shielded by a peak which is a solvent peak and has no influence on the technical content of the application.
Preparation Experimental example 1 (preparation of injection)
1000mg of the compound 1-10 obtained in the example 1 is taken, 1000ml of water for injection is respectively added, the pH value is adjusted to 7 by using sodium carbonate, the mixture is stirred to be dissolved, sterilized, filtered, filled and sealed, and the mixture is sterilized by flowing steam at 100 ℃ for 15 minutes to respectively prepare each injection containing 1mg/ml of the compound 1-10 for injection.
Formulation experimental example 2: (preparation of capsules)
The compound can be one or more of the compounds 1-10 prepared in example 1, in this example, the compound 3 is adopted, the compound 3 prepared in example 1 is weighed according to the weight of the formula, microcrystalline cellulose, sodium carboxymethyl starch and sodium dodecyl sulfate are respectively weighed and mixed, dry granulation is carried out by adopting a rolling method, then the mixture is uniformly mixed with magnesium stearate and filled into a No. 3 hollow capsule, and capsules containing 100 mg/capsule of the compound prepared in example are respectively prepared for oral administration.
Formulation experimental example 3: (preparation of tablets)
The compound can be one or more of the compounds 1-10 prepared in example 1, in the example, the compound 1 is adopted, the compound 1 obtained in example 1, starch, cross-linked PVP, sodium carboxymethyl starch and magnesium stearate are weighed according to the weight of the formula and mixed, 5% PVP-ethanol solution (the volume percentage of ethanol to water is 75: 25) is added to prepare soft materials, the soft materials are granulated through a 18-mesh sieve, after drying at 60 ℃ for 1h and 20-mesh size stabilization, talcum powder is added to the mixture after the whole grain is finished, the mixture is mixed uniformly and tabletted, and tablets with the specification of containing 1100 mg of the compound are prepared for oral administration.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A monoterpene bishydroxycoumarin compound shown as formula (I), and its enantiomer, diastereomer, cis-trans isomer, racemate, pharmaceutically acceptable salt or their mixture:
wherein, with R1、R2、R3And R4Connected by
Independently selected from a single bond or a double bond; the carbon atom with "#" and the carbon atom with "#" are chiral carbon atoms, the carbon atom with "#" is in R configuration, S configuration, or a combination thereof, and the carbon atom with "#" is in R configuration, S configuration, or a combination thereof;
X1、X2、X3and X4Independently selected from carbon atoms or oxygen atoms;
R1-R4independently selected from H, hydroxyl, oxygen atom, C1-C6Alkyl of (C)3-C6Cycloalkyl and C1-C6One of alkoxy groups of (a);
R5、R6independently selected from H,Hydroxy, C1-C5Alkyl of (C)3-C5Cycloalkyl and C1-C5One of the alkoxy groups of (a).
2. The monoterpene biscoumarin compound according to claim 1, which is an enantiomer, a diastereomer, a cis-trans isomer, a racemate, a pharmaceutically acceptable salt or a mixture thereof,
R1、R2、R3、R4independently selected from one of H, hydroxyl, oxygen atom, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, ethoxy, n-propoxy and isopropoxy;
when R is1、R2、R3Or R4When being an oxygen atom, bound to said oxygen atom
Is a double bond;
R5、R6independently selected from one of H, hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, methoxy, ethoxy, n-propoxy and isopropoxy.
3. The monoterpene biscoumarin compound according to claim 1 or 2, wherein X is selected from the group consisting of enantiomers, diastereomers, cis-trans isomers, racemates, pharmaceutically acceptable salts and mixtures thereof1And X2One of them is an oxygen atom and the other is a carbon atom; x3And X4One of them is an oxygen atom and the other is a carbon atom;
R1and R2One of them is an oxygen atom and the other is a hydroxyl group; r3And R4One is an oxygen atom and the other is a hydroxyl group;
X1and R1At the same timeIs an oxygen atom or is not an oxygen atom at the same time; x2And R2Simultaneously oxygen atoms, or simultaneously not oxygen atoms; x3And R3Simultaneously oxygen atoms, or simultaneously not oxygen atoms; x4And R4Either simultaneously with oxygen atoms or simultaneously without oxygen atoms.
4. The monoterpene bishydroxycoumarin compound according to any one of claims 1 to 3, which is an enantiomer, a diastereomer, a cis-trans isomer, a racemate, a pharmaceutically acceptable salt or a mixture thereof, wherein the monoterpene bishydroxycoumarin compound has the following structures (II) to (V):
5. the monoterpene bishydroxycoumarin compound according to any one of claims 1 to 4, which is a compound having a structure shown as follows, and enantiomers, diastereomers, cis-trans isomers, racemates, pharmaceutically acceptable salts or mixtures thereof:
6. a process for the preparation of a monoterpene bishydroxycoumarin according to any one of claims 1 to 5 which comprises the steps of:
1) extracting the trifoliate bitter stems with an organic solvent for one or more times to obtain an organic solvent extract;
2) dispersing the organic solvent extract with water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, and recovering ethyl acetate extract;
3) separating the ethyl acetate extract by a silica gel chromatographic column, carrying out gradient elution by using cyclohexane/ethyl acetate as an eluent to obtain a crude component, and carrying out one-time MCI column chromatography, one-time reverse ODS column chromatography and one-time Sephadex LH-20 column chromatography on the obtained crude component to obtain a column chromatography component containing the monoterpene dicoumarin compound;
4) separating and refining the column chromatography component containing the monoterpene bishydroxycoumarin compound by HPLC to obtain the monoterpene bishydroxycoumarin compound.
7. The method for producing a monoterpene bishydroxycoumarin compound according to claim 6, which further satisfies at least one of the following requirements (1) to (8);
(1) in the step 1), the organic solvent is at least one of n-hexane, dichloromethane, ethyl acetate, n-butanol, methanol, 50-95% methanol aqueous solution, ethanol and 50-95% ethanol aqueous solution;
(2) in the step 1), the extraction is reflux extraction or ultrasonic extraction, preferably reflux extraction, wherein the time of reflux extraction is 1-3h, and the times are 2-5;
(3) in the step 1), adding an organic solvent with 8-12 times of volume;
(4) in the step 1), the organic solvent extract is preferably an extract obtained by extracting trifurcate bitter stems and then evaporating to dryness;
(5) in the step 3), in the process of one-time MCI column chromatography separation, the column chromatography adopts a volume ratio of 2: 3 → 1: performing gradient elution by 0 methanol and water; the one-time reverse ODS column chromatography adopts a column chromatography with the volume ratio of 3: 2 → 0: 1, performing gradient elution by using methanol and water;
(6) in the step 3), in the separation process of the silica gel chromatographic column, the volume ratio of the silica gel chromatographic column is 100: 1 → 0: 1, taking cyclohexane and ethyl acetate as eluent for gradient elution;
(7) in the step 3), methanol or ethanol is adopted as an eluent for elution in the Sephadex LH-20 column chromatography separation process;
(8) in the step 4), acetonitrile or methanol water solution with the volume percentage of 50-75% is adopted as a mobile phase in the separation and refining process; after separation and refining, the solution is resolved by a chiral HPLC column.
8. A pharmaceutical composition comprising the monoterpene bishydroxycoumarin compound of any one of claims 1 to 5, an enantiomer, a diastereomer, a cis-trans isomer, a racemate, a pharmaceutically acceptable salt or a mixture thereof, and optionally a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier is selected from at least one of pharmaceutically acceptable solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, polymeric matrix materials, and film-forming materials;
the pharmaceutical composition is gel, cream, tablet, capsule, powder, mixture, pill, granule, solution, syrup, soft extract, suppository, aerosol, emplastrum, ointment, injection, spray, liniment, tincture, wet dressing, paste, lotion or sustained and controlled release preparation.
9. Use of a monoterpene biscoumarin compound according to any one of claims 1 to 5, an enantiomer, a diastereomer, a cis-trans isomer, a racemate, a pharmaceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition according to claim 8, for the preparation of an acetylcholinesterase inhibitor.
10. Use of the monoterpene biscoumarin compound of any one of claims 1 to 5, an enantiomer, a diastereomer, a cis-trans isomer, a racemate, a pharmaceutically acceptable salt thereof, or a mixture thereof, or the pharmaceutical composition of claim 8 for the preparation of a medicament for the prevention or treatment of a neurodegenerative disease, preferably senile dementia, memory impairment, brain tissue degenerative syndrome, or cholinergic neurodegeneration.
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| CN114028366A (en) * | 2021-10-30 | 2022-02-11 | 北京中医药大学东直门医院 | Application of monocyclic monoterpene compound in preparation of anti-senile dementia drugs |
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| US20110306605A1 (en) * | 2008-12-22 | 2011-12-15 | Sloan-Kettering Institute For Cancer Research | Coumarin-based compounds for the treatment of alzheimer's disease and cancer |
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| US20110306605A1 (en) * | 2008-12-22 | 2011-12-15 | Sloan-Kettering Institute For Cancer Research | Coumarin-based compounds for the treatment of alzheimer's disease and cancer |
| CN102274211A (en) * | 2011-06-17 | 2011-12-14 | 淮海工学院 | New purpose of coumarin compound |
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| CHOUDHURY M.HASAN等: "Chemical similarities between Microcybe multiflora turcz,and the rutaceous genus Phebalium", 《BIOCHEM.SYST.ECOL.》 * |
| CORINNE BRUHLMANN等: "Coumarins Derivatives as Dual Inhibitors of Acetylcholinesterase and", 《J.MED.CHEM.》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113754677A (en) * | 2021-08-18 | 2021-12-07 | 厦门大学 | A new coumarin dimer and its preparation method and application |
| CN113754677B (en) * | 2021-08-18 | 2022-09-20 | 厦门大学 | Novel coumarin dimer and preparation method and application thereof |
| CN114028366A (en) * | 2021-10-30 | 2022-02-11 | 北京中医药大学东直门医院 | Application of monocyclic monoterpene compound in preparation of anti-senile dementia drugs |
| CN114028366B (en) * | 2021-10-30 | 2023-02-28 | 北京中医药大学东直门医院 | Application of monocyclic monoterpene compound in preparation of anti-senile dementia drugs |
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| CN111848565B (en) | 2023-01-31 |
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