CN110204592B - Isopimarane diterpenoid compound and preparation method and application thereof - Google Patents
Isopimarane diterpenoid compound and preparation method and application thereof Download PDFInfo
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- CN110204592B CN110204592B CN201910483321.4A CN201910483321A CN110204592B CN 110204592 B CN110204592 B CN 110204592B CN 201910483321 A CN201910483321 A CN 201910483321A CN 110204592 B CN110204592 B CN 110204592B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/06—Antiarrhythmics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/006—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by sulfur as hetero atom
Abstract
Disclosed are isopimarane-type diterpene compounds having an anticonvulsant activity and an anti-hypoxic activity in myocardial cells. The compound is separated from amber and has the following structural formula. Proved by zebra fish convulsion model experiments, the isopimarane diterpenoid compound provided by the invention has an anticonvulsant effect; the in vitro anti-hypoxia injury activity shows that the isopimarane diterpenoid compound provided by the invention has the capacity of protecting myocardial cells against hypoxia.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an isopimarane diterpenoid compound and application thereof in preparing medicines for treating convulsion and cardiovascular diseases.
Background
Epilepsy was documented in the first 2000 b.c., and in the history of seizures in different human civilizations. As one of the common diseases in nervous system diseases, epilepsy has a second incidence rate in nervous system diseases than cerebrovascular diseases. There are about 7000 thousands of epileptic patients worldwide, and the occurrence of epileptic disease not only can cause health problems, makes the patient whole health condition low, intelligence and physical function impaired, the risk of accident and injury is higher, and a series of psychosocial problems such as mood, family, occupation that accompany epileptic production cause the huge influence to patient's whole quality of life. In addition, epilepsy is subject to significant direct medical costs and indirect costs due to loss of quality of life and productivity, creating a heavy financial burden.
Cardiovascular diseases, also known as circulatory diseases, refer to a series of diseases of the circulatory system; the circulatory system refers to the organs and tissues in the human body that carry blood, mainly including the heart, blood vessels (arteries, veins, microvessels), and can be subdivided into acute and chronic, and are generally associated with atherosclerosis. The most common cardiovascular diseases are arrhythmia, myocardial ischemia, angina pectoris, myocardial infarction, etc. Cardiovascular diseases are one of the leading causes of death in humans, and are also one of the diseases that consume enormous medical resources. The number of deaths worldwide from cardiovascular disease annually is about 1700 thousands. The prevalence rate of cardiovascular diseases in China is in a continuous rising stage, and the number of patients suffering from cardiovascular diseases is calculated to be 2.9 million in China cardiovascular disease report 2017, wherein 1300 million patients suffering from cerebral apoplexy, 1100 million patients suffering from coronary heart disease and 2.7 million patients suffering from hypertension are calculated.
Amber resin of ancient Pinus plant of Pinaceae is buried in underground for a long time to transform into resiniferous fossil. Has the main functions of tranquilizing and allaying excitement, promoting blood circulation and removing blood stasis, and inducing diuresis for treating stranguria. Can be used for treating palpitation, insomnia, convulsion, epilepsy, dysuresia, hematuria, and odynuria. According to the compendium of materia Medica records: has sweet and mild smell and no toxicity. The traditional Chinese medicine is mainly used for calming the five internal organs, calming soul, killing essence and evil demon, eliminating blood stasis, dredging five stranguria, strengthening heart, improving eyesight and grinding nebula, stopping heart pain and depressive psychosis, treating poison of venomous, breaking knots, treating postpartum blood occipital pain, stopping bleeding and promoting tissue regeneration. It is mainly used for nourishing heart, tranquilizing mind, removing blood stasis, promoting granulation, treating gynecological diseases, treating infantile abdominal mass, and treating nephelium with conjunctival congestion.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a new isopimarane diterpenoid compound, the application of which in the aspect of medicines for treating convulsion and cardiovascular diseases is screened out through a large amount of experiments, and the invention also aims to provide a preparation method thereof.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an isopimarane-type diterpene compound having a structure represented by the following formula (1):
the other technical scheme of the invention is as follows: a medicine for treating convulsion and cardiovascular disease comprises isopimarane diterpene compound shown in formula (1) or its salt.
Preferably, the pharmaceutical preparation is prepared from the isopimarane diterpenoid compound shown in the formula (1) and pharmaceutically acceptable pharmaceutical excipients into clinically applicable sprays, tablets, capsules, granules, pills, injections and the like.
Use of an isopimarane-type diterpene for the preparation of an anticonvulsant medicament, said neurological disorder comprising convulsions, epilepsy.
Use of an isopimarane diterpene for the preparation of a medicament for the treatment of cardiovascular diseases, including angina pectoris, myocardial infarction, myocardial ischemia, heart failure and arrhythmia. Particularly preferred cardiovascular disease is myocardial ischemia.
The preparation method of the isopimarane diterpenoid compound provided by the invention comprises the following steps:
(1) pulverizing Succinum, extracting with ethyl acetate, and concentrating the extractive solution to obtain ethyl acetate part;
(2) separating the ethyl acetate part obtained in the step (1) by adopting an AB-8 macroporous resin column chromatography, eluting with a 50% methanol solution, then eluting with methanol, collecting methanol eluent, and recovering the methanol to obtain a methanol elution part;
(3) and (3) dissolving the methanol elution part obtained in the step (2) with acetonitrile, and separating by adopting a high-pressure preparation liquid phase to obtain the isopimarane diterpenoid compound.
As a preferable mode, the preparation method of the isopimarane diterpenoid compound provided by the invention comprises the following steps:
(1) taking 100g of amber, crushing, extracting twice with 10 times of ethyl acetate for 2 hours each time, combining ethyl acetate extracting solutions, and recovering ethyl acetate to obtain an ethyl acetate part;
(2) performing AB-8 macroporous resin column chromatography on the ethyl acetate part obtained in the step (1), eluting 3 column volumes by 50% methanol solution, eluting 5 column volumes by methanol, collecting methanol elution part, and recovering methanol to obtain methanol part;
(3) and (3) dissolving the methanol part obtained in the step (2) by using acetonitrile with the volume concentration of 40%, and separating by adopting a high-pressure preparation liquid phase to obtain the isopimarane diterpenoid compound.
The preferred high pressure preparation conditions are: SEPAX GP-C18(10X 250mm,5 μm) column, 40% acetonitrile isocratic elution, flow rate of 3mL/min, detection wavelength 227 nm.
In the above preparation methods, the extraction method in step (1) may be cold soaking, percolation, microwave extraction, ultrasonic extraction, reflux extraction, etc.
Has the advantages that:
the invention carries out systematic and intensive research on chemical components of the amber medicinal material, and shows that an isopimarane diterpene is separated from the amber medicinal material through spectrum, mass spectrum and x single crystal diffraction analysis and is a new compound through retrieval.
In-vivo and in-vitro activity researches show that the isopimarane diterpenoid provided by the invention has obvious anti-epilepsy and convulsion effects, and also has a good protection effect on ischemic myocardial cells.
Toxicity experimental study shows that the isopimarane diterpenoid compound with anti-epilepsy and anti-myocardial ischemia activities provided by the invention has low toxicity and good safety.
Drawings
FIG. l is a schematic diagram of the structure of succinic neo-acid.
FIG. 2 is a scheme of succinic acid1HNMR spectrogram.
FIG. 3 is a scheme of succinic acid13C NMR spectrum.
FIG. 4 is a graph of the inhibition of pentaerythrine-induced convulsions in zebrafish by succinic acid; in the figure, A is the shortened swimming distance, and B is the reduced high speed swimming rate.
FIG. 5 is a graph showing that succinate increases the anti-hypoxic ability of H9C 2.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
Example 1 preparation of an isopimarane-type diterpene compound comprising the following steps:
(1) 100g of amber was taken, pulverized, and extracted twice with 10 times of ethyl acetate for 2 hours each time, and ethyl acetate was recovered to obtain an ethyl acetate fraction.
(2) Taking the ethyl acetate part obtained in the step (1), performing AB-8 macroporous resin column chromatography, eluting by 50% methanol solution for 3 column volumes, eluting by methanol for 5 column volumes, collecting ethanol elution part, and concentrating;
(3) and (3) re-dissolving the methanol part obtained in the step (2) with 40% acetonitrile, and separating by adopting a high-pressure preparation liquid phase to obtain an isopimarane diterpenoid compound (named as succinic neo-acid), wherein the structural formula is shown in figure 1. The specific conditions for preparing the liquid phase under high pressure are as follows: : SEPAX GP-C18(10X 250mm,5 μm) column, 40% acetonitrile isocratic elution, flow rate of 3mL/min, detection wavelength 227 nm.
Structural analysis of succinic neo-acid: needle crystals (acetonitrile). The ultraviolet spectrum has a characteristic absorption at λ max 227 nm. HR-ESI-MS M/z 353.2153[ M + H ]]+(calcd for C20H33O3S+，353.2150；Δ＝0.85ppm)，Ω＝6。
As shown in figure 2 of the drawings, in which,1H-NMR(400MHz,DMSO-d6) The spectrum shows three methyl signals delta in the high field regionppm 1.10(3H,s,H-17)，1.08(3H,s,H-19)，0.86(3H,s,H-20)；
As shown in figure 3 of the drawings,13C-NMR(100MHz,DMSO-d6) Spectral and DEPT spectral information revealed that the compound contained 20 carbons, including 3 methyl carbons: deltappm23.1(C-17), 16.8(C-19) and 0.86 (C-20); 8 methylene carbon number deltappm37.9(C-1), 17.6(C-2), 23.8(C-6), 33.2(C-7), 19.7(C-11), 32.0(C-12), 40.4(C-15) and 48.5 (C-16); 5 methine carbons dppm46.3(C-4), 48.9(C-5), 32.6(C-8), 51.1(C-9) and 84.2 (C-14); 3 quaternary carbons: deltappm44.80(C-4), 35.8(C-10) and 42.6 (C-13); 1 carbonyl carbon: deltappm180.4(C-18) and a sulfinyl group.
Diffraction on X single crystal:
on a bruker apex-II CCD diffractometer, MoK α rays (wavelength λ ═ 0.71073a) monochromatized with a graphite monochromator were used as incident radiation. At 2.01<θ<In the range of 27.50deg., in a w-20 scanning mode, 18426 reflection points are acquired under 296(2) K, wherein 4482 are independent observable points [ I>2\s(I)]R (int) ═ 0.0305, all intensity data were corrected using the SADABS program. The crystal belongs to an orthorhombic system, and the space group is Pnma, a is 11.2366(8), b is 11.4228(8), c is 14.8442(10), alpha is 90deg., beta is 90deg., gamma is 90deg., V is 1905.3(2), Z is 4, Dc is 1.229Mg/m3. The crystal structure is solved by a direct method (SHELXL-97), all non-hydrogen atoms are obtained through multi-round Fourier synthesis, and the hydrogen atoms are obtained by direct hydrogenation after theoretical calculation. And correcting all non-hydrogen atom coordinates and anisotropic thermal parameters to be convergent by adopting a full matrix least square method. The final bias factors are R ═ 0.0287 and wR ═ 0.0800. The molecular structure diagram was plotted using the SHELXl-97 package.
Example 2 zebrafish anticonvulsant experiments
The AB strain zebra fish is divided into 6 groups by experimental grouping and administration: control group (Control), model group (pentaerythrite intervention), positive drug group (phenobarbital, PB), 3 concentration (10, 5, 1 μ M) dose groups of the succinic acid compound prepared in example 1, each group of 10 juvenile fishes with 4-7dpf, each drug group is given drug for 72 hours first, then 6mM pentaerythrite intervention is carried out for 10 minutes, then 10 minutes of video recording is collected, image processing is carried out by utilizing Matlab 2018a self-programming program, and information such as total swimming distance, speed, swimming area distribution and the like is counted.
The statistical method comprises the following steps: calculate data to
Statistical analysis was performed using SPSS statistical software, single-factor analysis of variance was used for the comparisons between groups, and T-test was used for the comparisons between both groups, with a test level p of 0.05.
As shown in fig. 4: fig. 4A shows that the total swimming distance of the zebra fish can be remarkably shortened by the succinic acid at the dosage of 5 mu M and 10 mu M, and fig. 4B shows that the proportion of the zebra fish swimming at high speed can be remarkably reduced. (Control group Control, model group, group of positive drugs PB, and low dose group of succinic acid Compound C3N11 μ M, dose group C in the succinic neo-acid Compound3N15 μ M, high dose group C of succinic neo-acid compounds3N1-10μM)
The experimental results show that the novel compound succinic acid obtained by separation has good convulsion resisting effect.
Example 3 anti-cardiomyocyte H9C2 hypoxia assay
Rat cardiomyocytes (H9C2) were selected. Experimental groups and dosing were divided into 8 groups: blank and model groups (600 μm CoCl)2Intervention), 6 concentration (50, 15, 5, 1.5, 0.5, 0.15 μm) dose groups of the succinic neo-acid compound prepared in example 1. The drugs in each drug group were administered for 48 hours first, and then CoCl was added2And intervening for 24 h. Cell activity was measured by MTT method.
The statistical method comprises the following steps: data were calculated to show that statistical analysis was performed using SPSS statistical software, single factor analysis of variance was used for the comparisons between groups, T-test was used for the comparisons between two groups, and the test level p was 0.05.
As shown in fig. 5, the experimental results: the 5-50 mu M concentration of the neosuccinic acid compound can improve the survival rate of rat myocardial cells under the condition of hypoxia H9C 2. Particularly, the survival rate of rat myocardial cells under the condition of hypoxia H9C2 can be obviously improved when the concentration is 015 mu M. P < 0.05.
The experimental results show that the neosuccinic acid separated by the invention has the effect of protecting the myocardial cells.
Claims (8)
1. An isopimarane-type diterpene compound having an anticonvulsant activity and an antihypoxic activity for protecting cardiac muscle cells, characterized in that the structural formula thereof is:
2. a medicament for treating epilepsy, convulsive disorders and cardiovascular disorders, comprising the isopimarane-type diterpene compound or a salt thereof according to claim 1.
3. The medicament of claim 2, wherein the dosage form is spray, tablet, capsule, granule, pill, or injection.
4. The process for producing an isohimarane-type diterpene compound according to claim 1, which comprises the steps of:
(1) pulverizing Succinum, extracting with ethyl acetate, and concentrating the extractive solution to obtain ethyl acetate part;
(2) separating the ethyl acetate part obtained in the step (1) by adopting an AB-8 macroporous resin column chromatography, eluting with a 50% methanol solution, then eluting with methanol, collecting methanol eluent, and recovering the methanol to obtain a methanol elution part;
(3) dissolving the methanol elution part obtained in the step (2) with acetonitrile, and separating by adopting a high-pressure preparation liquid phase to obtain an isopimarane diterpenoid compound; the high-pressure preparation liquid phase separation conditions are as follows: SEPAX GP-C18Specification 10X 250mm,5 μm chromatographic column, 40% acetonitrile isocratic elution, flow rate of 3mL/min, detection wavelength 227 nm.
5. The method for producing an isopimarane-type diterpene compound according to claim 4, wherein the extraction method of step (1) is cold leaching, percolation, microwave extraction, ultrasonic extraction or reflux extraction.
6. Use of the isopimarane-type diterpene compound according to claim 1 for the preparation of an anti-epileptic, anticonvulsant drug.
7. Use of the isopimarane-type diterpene compound according to claim 1 for the preparation of a medicament for the treatment of cardiovascular diseases.
8. The use according to claim 7, wherein the cardiovascular disease is angina pectoris, myocardial infarction, myocardial ischemia, heart failure and arrhythmia.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103360351A (en) * | 2013-07-17 | 2013-10-23 | 云南大学 | Isopimarane diterpenoid compounds and application thereof |
| CN107021942A (en) * | 2016-02-01 | 2017-08-08 | 复旦大学 | Bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and the purposes in pharmacy |
| CN108530281A (en) * | 2018-06-11 | 2018-09-14 | 云南大学 | A kind of isopimarane type forskolin, medical composition and its use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103360351A (en) * | 2013-07-17 | 2013-10-23 | 云南大学 | Isopimarane diterpenoid compounds and application thereof |
| CN107021942A (en) * | 2016-02-01 | 2017-08-08 | 复旦大学 | Bark extract of pinus fenzeliana var dabeshanensis and preparation method thereof and the purposes in pharmacy |
| CN108530281A (en) * | 2018-06-11 | 2018-09-14 | 云南大学 | A kind of isopimarane type forskolin, medical composition and its use |
Non-Patent Citations (1)
| Title |
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| 大百合果实中的异海松烷型二萜化合物;刘润民;《云南植物研究》;19840701;第6卷(第2期);第219-222页 * |
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