CN113372355B - Hexahydrobenzophenanthridine alkaloid and preparation method and application thereof - Google Patents
Hexahydrobenzophenanthridine alkaloid and preparation method and application thereof Download PDFInfo
- Publication number
- CN113372355B CN113372355B CN202110522481.2A CN202110522481A CN113372355B CN 113372355 B CN113372355 B CN 113372355B CN 202110522481 A CN202110522481 A CN 202110522481A CN 113372355 B CN113372355 B CN 113372355B
- Authority
- CN
- China
- Prior art keywords
- compound
- hexahydrobenzophenanthridine
- alkaloid
- alkaloids
- extract
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
-
- 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
-
- 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
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
Landscapes
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Psychology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Epidemiology (AREA)
Abstract
The invention provides hexahydro benzophenanthridine alkaloid and a preparation method and application thereof, belonging to the technical field of biological medicines. The invention firstly uses Ardisia japonica Makino as a raw material, obtains two high-purity hexahydrobenzophenanthridine alkaloids by extraction, separation and purification, and tests prove that the hexahydrobenzophenanthridine alkaloids have an inhibition effect on thrombus caused by arachidonic acid, an inhibition effect on zebra fish inflammation caused by copper sulfate and a strong protection effect on dopamine neurons, so that the alkaloid compounds can be novel and effective medicaments for treating the heart Parkinson disease and have good practical application value.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to hexahydrobenzophenanthridine alkaloids as well as a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Ardisia japonica (Dactylicapnos torulosa) is a poppy family purple plant, an annual herb, and currently, only found in the southwest, the west, the middle to the southeast of Yunnan, the west of Guizhou, the southwest of Sichuan, and the southeast of Tibet in China. Under natural state, the plant grows under forests, bushes or ditch edges and roadside with the altitude of 1-3 000m. The main active ingredients in the ardisia crenata sims contain alkaloids, and have the activities of regulating cardiovascular system, relieving pain, relieving spasm, protecting liver, resisting anoxia, resisting malaria, etc. The ardisia japonica has complex chemical components and difficult active component separation, and few reports exist for separating specific high-purity hexahydrobenzophenanthridine alkaloids.
Inflammation also plays a major role in many epidemic conditions, such as alzheimer's disease, parkinson's, liver and lung cancer. In developed or developing countries, the neurodegenerative disease Parkinson's Disease (PD), caused by inflammation, is a serious threat to human health and human well-being. Parkinson's disease is one of the extrapyramidal diseases which are mostly caused in middle-aged and elderly people, is also called paralysis agitans, is the second major nervous system disease which is second to Alzheimer's disease at present, has a chronic progressive disease course, has no effective prevention method, and has the morbidity which is increased by times along with the increase of the age.
It has been reported that protopine, a component of dactylicapnos scandens, inhibits the 5-hydroxytryptamine transporter and the norepinephrine transporter and has an antidepressant effect in a mouse model, suggesting that they may improve neurodegenerative disease activity. Promotopine also has an effect on the release of platelet active substances that are affected by platelet aggregation, suggesting possible antithrombotic activity. The inventor finds that the predecessor only carries out systematic research on pain easing and cardiovascular effects of dactylicapnos. The benzophenanthridine alkaloid compounds in the ardisia crenata sims and the medical biological activity thereof are rarely reported.
Disclosure of Invention
Based on the defects of the prior art, the hexahydrobenzophenanthridine alkaloids and the preparation method and application thereof are provided by the invention, the ardisia crenata is taken as a raw material for the first time, two high-purity hexahydrobenzophenanthridine alkaloids are obtained through extraction, separation and purification, and experiments prove that the hexahydrobenzophenanthridine alkaloids have an inhibition effect on thrombus caused by arachidonic acid, have an inhibition effect on zebra fish inflammation caused by copper sulfate and have a strong protection effect on dopamine neuron, so that the alkaloid compound can be a novel and effective medicine for treating the heart Parkinson disease and has a good value in practical application.
In a first aspect of the invention, there is provided a compound of formula (I):
wherein R may be selected from OCH 3 Or H;
when R is selected from OCH 3 Then, compound 1:4-methoxychelidonine;
when R is selected from H, the compound is the compound 2: chelidonine.
In some embodiments of the invention, hexahydrobenzophenanthridine alkaloids of formula (I) may be prepared in crystalline or amorphous form, and if crystalline, may optionally be solvates, for example as hydrates.
In a second aspect of the present invention, there is provided a process for the preparation of the above compound, which comprises:
s1, adding ethanol into ardisia japonica, soaking and extracting, and combining concentrated extracting solutions to obtain a crude extract;
s2, suspending the crude extract in diluted hydrochloric acid, performing suction filtration to obtain a filtrate, adjusting the pH to be alkaline, and sequentially extracting by using petroleum ether, dichloromethane and n-butanol;
s3, separating the dichloromethane extract by a silica gel column, performing gradient elution by using dichloromethane-methanol with a gradient of 200 to 1;
and S4, treating the n-butanol extract by an MCI column, performing gradient elution by using methanol-water with a gradient of 1.
In a third aspect of the invention there is provided the use of a compound as described above in any one or more of:
a) Inhibiting thrombosis and/or preparing medicines related to inhibiting thrombosis;
b) Inhibiting inflammatory reaction and/or preparing medicaments related to the inhibition of inflammatory reaction;
c) (ii) protection of dopamine neurons and/or protection of dopamine neuron-related drugs;
d) Treating heart Parkinson disease and/or preparing medicine for treating heart Parkinson disease.
In a fourth aspect of the invention, there is provided a method of treating cardiac parkinson's disease, said method comprising administering to a subject a therapeutically effective amount of a compound as described above.
The beneficial technical effects of one or more technical schemes are as follows:
in the technical scheme, the two hexahydrobenzene phenanthridine alkaloids are simultaneously separated and efficiently obtained from the ardisia japonica, so that the comprehensive utilization of natural medicine resources is realized, and an effective way is provided for searching new active ingredients of medicines.
Meanwhile, through the test experiment and the structure-activity relationship research on the anti-inflammatory, antithrombotic and MPTP-induced Parkinson activity of the alkaloid compound separated from ardisia japonica, the result shows that hexahydrobenzophenanthridine alkaloid has an inhibition effect on the thrombus caused by arachidonic acid, has an inhibition effect on the inflammation of zebrafish caused by copper sulfate and has a strong protection effect on dopamine neurons, and the alkaloid compound is possibly a novel and effective medicament for treating the cardiac Parkinson disease, so that the alkaloid compound has a good practical application prospect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a HR-ESI-MS spectrum of 4-methoxycyclidine isolated by an example of the present invention.
FIG. 2 shows 4-methoxyhedgeanine isolated by the present invention 1 HNMR atlas (600MHz, in CD 3 OD)。
FIG. 3 shows 4-methoxycholedoni isolated by an embodiment of the present inventionOf ne 13 CNMR atlas (400MHz, in CD 3 OD)。
FIG. 4 is an HSQC spectrum (400MHz, in CD) of 4-methoxyhedonine isolated in the examples of the present invention 3 OD)
FIG. 5 is an HMBC profile of 4-methoxychelidonine isolated in accordance with an embodiment of the present invention (400MHz, incD) 3 OD)。
FIG. 6 shows (+) -chelidonine isolated from an embodiment of the invention 1 HNMR map (600MHz, in CDCl 3 )。
FIG. 7 shows (+) -chelidonine isolated according to an embodiment of the invention 13 CNMR atlas (400MHz, in CDCl 3 )。
FIG. 8 shows the results of experiments conducted by compounds 1 and 2 of the present invention at 3 different concentrations to inhibit inflammation induced by copper sulfate in zebra fish; wherein, a is an experimental map of inflammatory response, and b is a statistical map of experimental data.
FIG. 9 shows the results of the determination of antithrombotic activity of compounds 1 and 2 of the examples of the present invention at 3 different concentrations; wherein, a is an actual map of antithrombotic activity, and b is a statistical map of test data.
FIG. 10 is the results of dopamine neuron protection assays for compounds 1 and 2 according to examples of the present invention; wherein a is a length statistical chart of each group of DA neurons; b is a diagram of the condition of DA neurons of the juvenile fish observed by each group of fluorescence microscopes; and c is a motion trail diagram of each group of zebra fishes.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, the benzophenanthridine alkaloid compounds and their pharmaceutical biological activities in Ardisia japonica are only reported at present.
In view of the above, in one exemplary embodiment of the present invention, there is provided a benzophenanthridine alkaloid compound represented by formula (I):
wherein R may be selected from OCH 3 Or H;
when R is selected from OCH 3 When this is compound 1:4-methoxychelidonine;
when R is selected from H, the compound is the compound 2: chelidonine.
In some embodiments of the invention, hexahydrobenzophenanthridine alkaloids of formula (I) may be prepared in crystalline or amorphous form, and if crystalline, may optionally be solvates, for example as hydrates.
In another embodiment of the present invention, there is provided a method for preparing the above compound, comprising:
s1, adding ethanol into ardisia japonica, soaking and extracting, and combining concentrated extracting solutions to obtain a crude extract;
s2, suspending the crude extract in diluted hydrochloric acid, performing suction filtration to obtain a filtrate, adjusting the pH to be alkaline, and sequentially extracting by using petroleum ether, dichloromethane and n-butanol;
s3, separating the dichloromethane extract by a silica gel column, performing gradient elution by using dichloromethane-methanol with gradient of 200 to 1;
and S4, treating the n-butanol extract by an MCI column, performing gradient elution by using methanol-water with a gradient of 1.
The extraction preparation method gradually separates high-purity monomer components of the compound from the ardisia japonica in a coarse-fine mode.
The gradient elution in the present invention refers to that the concentration ratio of the mobile phase is changed continuously during the elution process, but the initial concentration and the final concentration are fixed, for example, "gradient elution is performed using dichloromethane-methanol with a gradient of 200: the dichloromethane-methanol was subjected to gradient elution in such a manner that the starting volume ratio was 200 and the elution end volume ratio was 1.
The individual steps of the preparation process according to the invention can be further modified, for example:
in still another embodiment of the present invention, in the step S1,
in the step S1, the raw materials of the ardisia japonica comprise an underground part and an overground part of the ardisia japonica;
the solvent and feed liquid ratio used in extraction have influence on the impurity content of crude extract and the extraction rate of effective components. The solvent is preferably a 90 to 95% ethanol solution, and more preferably a 95% ethanol solution. The feed liquid ratio of the ardisia japonica and the ethanol is 1kg: 2-7L (preferably 1.06kg. More specifically, the leaching is carried out by a multiple leaching process. The extraction conditions are adopted to effectively reduce the content of impurities in the crude extract, thereby further improving the extraction rate of hexahydrobenzophenanthridine alkaloids.
In still another embodiment of the present invention, in the step S2,
the dilute hydrochloric acid is 0.5-5% of dilute hydrochloric acid, and the concentration of the hydrochloric acid is controlled to be 0.5%, 1%, 2%, 3% or 5%;
in the step of adjusting the pH to be alkaline, the pH of the filtrate is adjusted to 9-12 by using 20-30% ammonia water.
The specific method for sequentially extracting by using petroleum ether, dichloromethane and n-butanol comprises the following steps:
adding petroleum ether, standing and extracting for 10-30 min, collecting supernatant, namely extracting solution, recovering the petroleum ether under reduced pressure at 40-50 ℃, using the recovered petroleum ether for extraction again until the color of the finally obtained extracting solution is light green, and recovering the petroleum ether under reduced pressure to obtain petroleum ether extract; sequentially extracting dichloromethane and n-butanol according to the extraction process of petroleum ether to obtain dichloromethane extract and n-butanol extract respectively;
in still another embodiment of the present invention, in the step S3,
the specific procedure of gradient elution with dichloromethane-methanol is as follows: volume ratio of dichloromethane to methanol =200, 1, 180; by adopting the optimized gradient condition, the separation degree can be improved;
further, TLC thin-layer chromatography and mass spectrum tracking detection are adopted, fractions mainly containing target components are collected and concentrated, the obtained extract silica gel thin-layer detection is carried out, and similar components are combined to obtain 8 components, namely S1-S8; the S3 fraction was subjected to liquid chromatography (1.5 mL/min, meOH-H) 2 O (0.1-tea) 77) to obtain the compound 1, i.e., 4-methoxycyclidine (t) R ＝28.0min)；
The liquid chromatography parameters were as follows:
chromatograph: agilent 1260-DAD;
and (3) chromatographic column: agilent YMC-Pack ODS-AS-5 μm,12nm (250 mm. Times.10 mm) -C18;
the eluent is: mobile phase B methanol, mobile phase a 0.1% triethylamine water, flow rate: 1.5mL/min of the reaction solution,
isocratic elution: 0 to 15min, B:77vol%, A:23vol%.
The compound 1 with high purity can be directly obtained by adopting the liquid chromatography separation.
In still another embodiment of the present invention, in the step S4,
performing gradient elution by using methanol and water, wherein the specific procedure of the gradient elution is as follows: methanol to water volume ratio =1, 2, 3, 2, 4.
Further, fractions mainly containing the target component were collected by TLC thin layer chromatography and mass spectrometric follow-up detection to obtain 4 fractions (3I 1 to 3I 4). 3I4 gradient elution with methanol and water as eluent (V/V = 3; separation by ODS column yielded 9 fractions (3I 4O1 → 3I4O 9). The compound 2, i.e., chelidonine, is obtained by recrystallization from 3I4O 5.
In a further embodiment of the invention, there is provided the use of a compound as described above in any one or more of:
a) Inhibiting thrombosis and/or preparing drugs related to inhibiting thrombosis;
b) Inhibiting inflammatory reaction and/or preparing medicaments related to the inhibition of inflammatory reaction;
c) (ii) protection of dopamine neurons and/or protection of dopamine neuron-related drugs;
d) Treating cardiac Parkinson's disease and/or preparing medicine for treating cardiac Parkinson's disease.
In yet another embodiment of the present invention, there is provided a method of treating cardiac parkinson's disease, said method comprising administering to a subject a therapeutically effective amount of a compound as described above.
The subject of the present invention refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
A therapeutically effective amount according to the present invention is that amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which response includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.
The range of therapeutically effective amounts that can be used will be known to the researcher, veterinarian, medical doctor or other medical professional in the art based on clinical trials or other means known in the art.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples extraction separation experiments
1) Twisting fruit ardisia japonica raw materials (10.6 Kg: extracting 1.1Kg of root and 9.5Kg of aerial part with 95% ethanol (3 × 30L, first 7 days, second 10 days, and third 3 days) by cold liquid immersion, mixing the three extractive solutions, and concentrating the extractive solution with large rotary evaporator (55 deg.C in water pot, vacuum degree of 55 deg.C, of Zyguyi City Rihua instruments, inc.)-0.06 Mpa) to obtain 500g of crude extract. Suspending part of the crude extract (483.2 g) in 1% diluted hydrochloric acid (3L), suction filtering to obtain filtrate, adjusting pH of the filtrate to 10 with 25% ammonia water, sequentially extracting with petroleum ether (3 × 3L) to obtain PE extract (1.7 g), and extracting with CH 2 Cl 2 (800mL, 700mL,800mL and 700 mL) to give CH 2 Cl 2 Extract (3.9 g), using n-BuOH (400 mL, 300mL and 300 mL) to give nBuOH extract (5.6 g), the remaining aqueous layer fraction (140.3 g).
Specifically, the extraction steps using petroleum ether, dichloromethane and n-butanol include: adding petroleum ether, standing and extracting for 10-30 min, collecting supernatant, namely extracting solution, recovering the petroleum ether under reduced pressure at 55 ℃, reusing the recovered petroleum ether for extraction until the color of the finally obtained extracting solution is light green, and recovering the petroleum ether under reduced pressure to obtain petroleum ether extract; and sequentially extracting dichloromethane and n-butanol according to the extraction process of petroleum ether to obtain dichloromethane extract and n-butanol extract respectively. The contents of the ardisia japonica extract are complex, and the effective components are primarily separated and enriched by adopting organic solvents with sequentially increased polarities.
2) Mixing the dichloromethane extract (3.7 g) and silica gel, loading the mixture into a column, performing gradient elution by a dichloromethane-methanol system, wherein the dichloromethane-methanol volume ratio is =10, 1, 5 and 1, simultaneously performing TLC (thin layer chromatography) and mass spectrum tracking detection, collecting fractions mainly containing target components, concentrating, and performing liquid chromatography separation on the obtained extract. Gradient V/V = 200. And S3, separating by adopting high performance liquid chromatography.
Liquid chromatography parameters: chromatograph: agilent 1260-DAD;
a chromatographic column: agilent YMC-Pack ODS-AS-5 μm,12nm (250 mm. Times.10 mm); the eluent is: mobile phase B is methanol, and mobile phase A0.1% (mass percent) of triethylamine water, flow rate: 1.5mL/min, isocratic elution: 0 to 15min, B:77vol%, A:23vol%. S3 can be separated by liquid chromatography to directly obtain high-purity 4-methoxyehelidonine (t) R ＝28.0min)。
3) The nBuOH extract (5.6 g) was passed through an MCI column, eluted successively with methanol and water, followed by detection by TLC thin layer chromatography and mass spectrometry, to collect a fraction mainly containing the objective component, followed by separation with methanol and water as an eluent through an ODS column, and then by recrystallization to obtain Compound-chelidonine.
Specifically, a methanol-water system is adopted for gradient elution, the volume ratio of methanol to water is = 1. Gradient elution was performed on 3I4 using reverse silica gel as a packing material (50 × 300 mm) and a methanol-water system as an eluent (V/V =3: 1), one fraction per 100mL of eluent, a total of 165 fractions were collected, similar fractions were combined according to TLC and uv-coloration (254 nm or 365 nm), and the solvent was recovered under reduced pressure to give a total of 9 fractions (3I 4O1 to 3I4O 9). 3I4O5 recrystallization to obtain (+) -chelidonine.
Effect verification
Experimental example 1:
the experiment establishes an inflammation model of the fluorescent transgenic zebra fish (Tg: zlyz-EGFP) stimulated by copper sulfate, counts the migration number of macrophages, screens the anti-inflammatory activity of hexahydrobenzophenanthridine alkaloid compounds with the general formula (I), and observes whether the compounds separated from Ardisia japonica have the potential of resisting inflammation.
Anti-inflammatory activity assay:
the activity test method is as follows:
healthy inflammatory cell fluorescent transgenic zebra fish fertilized with 72hpf is taken as an experimental animal and randomly divided into a sample treatment group, a model group, a blank control group and a positive control group (20 mu M ibuprofen) to be tested with the concentration of 5, 10 and 25 mu M, and each group is respectively added with ibuprofenGroups 10 embryos, while setting 2 replicate wells, each concentration is replicated three times. All the groups are put into a constant-temperature incubator at 28.5 ℃ for co-culture for 2h. Then using 20 mu M CuSO 4 The zebra fish of each sample group and the positive control group are respectively treated for 1h. After the treatment is finished, the zebra fish is cleaned, then the inflammatory reaction is observed under a fluorescence microscope, the number of inflammatory cells migrating to the zebra fish measuring line is calculated, and the GraphPad software statistically analyzes each group of data.
The results of the activity assay are shown in FIG. 8.
Among alkaloids, compound 2 has the best anti-inflammatory activity; the number of the Proepoxy methylene and the methoxyl and the position of the methoxyl influence the activity of the compound, and the methoxyl at the 4-position can weaken the activity of the compound; when the benzophenanthridine is 25 mu M, the anti-inflammatory activity is as follows: 2 >.
Experimental example 2:
the experiment establishes a thrombus model of wild zebra fish (AB) stimulated by arachidonic acid, counts the red dyeing area of the heart, screens the antithrombotic activity of hexahydrobenzophenanthridine alkaloid compounds with the general formula (I), and observes whether the compound separated from ardisia crenata has the antithrombotic potential.
Antithrombotic activity test:
the activity test method is as follows:
taking 72hpf wild type zebrafish embryos in a culture dish, selecting the embryos which are normally developed under a stereomicroscope, transferring the embryos into a 24-well culture plate, wherein 10 embryos are contained in each well, and each concentration is 2 multiple wells. Setting experimental groups, namely a solvent control group and a model group: adding 0.4% DMSO solution, 6h later, changing to 80 μ M AA solution; positive control group: adding ASP solution to make the final concentration 22.5 μ g/mL, placing in 28 deg.C incubator for 6h, and changing to AA solution with final concentration of 80 μ M; drug intervention group: adding the drug mother liquor to make the final concentration 5, 10 and 50 μ M, placing in 28 deg.C incubator for 6h, and changing to 80 μ M AA solution. Then, the mixture was placed in an incubator at 28 ℃ for 1.5 hours. Taken out, and stained with 1mg/mL o-dianisidine staining solution for 10min in the dark. Washing fish with water for 3 times, and taking pictures.
The results of the activity assay are shown in FIG. 9.
As seen from table 1, the positive control group (aspirin) had a thrombus prevention rate of 67.4%; the antithrombotic activities of the compounds 1 and 2 showed concentration dependency, and the thrombus-preventing rate of the compound 2 at 10. Mu.M was 76.5%.
TABLE 1 prevention of thrombosis rates for Compounds 1 and 2
Experimental example 3: experiment of the protective action of benzophenanthridine alkaloid compound with general formula (I) on dopamine neuron
1) Preparation of zebra fish embryos: when eggs are collected, firstly selectively mature zebra fish are separately placed into an egg laying tank with a partition plate at 5-6 pm according to the male-female ratio of 1; the next morning 8:30, extracting the partition plate, collecting fertilized eggs after 2 hours, repeatedly washing the fertilized eggs with fresh fish culture water for 3 times, transferring the fertilized eggs into fresh culture water containing 2mg/L methylene blue, and then placing the fertilized eggs in an incubator at 28 ℃ for incubation.
2) Experimental grouping and treatment: firstly, transgenic zebra fish Vmat at GFP of 1dpf after fertilization and wild zebra fish AB embryo are subjected to membrane removal treatment by using 1mg/mL Pronase E 131 Then cleaning the fish with fresh fish culture water for 3 times, putting the fish into a 6-hole plate, and setting the following experimental groups for 30 juvenile fishes in each hole: 5mL of a control group treated with fresh fish-farming water, 5mL of a group treated with MPTP at a concentration of 50. Mu.M, and 5mL of a group co-treated with MPTP at different concentrations (5. Mu.M, 10. Mu.M, 50. Mu.M) of the compound of Table 4.2 and 50. Mu.M were added (the compound and MPTP were added simultaneously to fresh fish-farming water to respective working concentrations). GFP in transgenic zebra fish is dosed with fresh fish-farming water containing 0.03mg/mL of PTU, which can inhibit melanin generation and facilitate observation of DA neurons. Adding medicine, culturing in 28 deg.C incubator, and changing the medicine every 24 hr.
3) Observation of DA neurons: at 4dpf, randomly selecting 8 juvenile fishes from different experimental groups of transgenic zebra fish Vmat: GFP, observing the DA neurons of the juvenile fishes by using a Zeiss stereomicroscope respectively, and taking pictures for recording.
4) Behavioral experiments: at 5dpf, randomly selecting 8 juvenile fishes from different experimental groups of wild zebra fish AB, putting the juvenile fishes into a 48-hole plate, putting one juvenile fish in each hole, adding 1mL of fresh fish culture water, putting the 48-hole plate into a dark box of a Zebrabox zebra fish behavior analyzer for 15min, and starting to perform behavioral detection after the juvenile fishes adapt to the environment. Zeblab software is used for collecting the motion trail of the zebra fish within 20min, and the software is used for exporting data and calculating the total distance.
The ratio of the length of the DA neuron region to the control for the positive drug at 10. Mu.M was 1.29, and the ratios of compound, 1 (5. Mu.M), 1 (10. Mu.M) and 2 (10. Mu.M) were 1.41,1.28 and 1.21, respectively. Among them, the DA protection of the novel compound 1 (5. Mu.M) is the strongest.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (1)
1. The application of the compound chelidonine in preparing medicines related to inhibiting thrombosis; the chemical compound chelidonine has a structural formula:
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211318400.8A CN115518069B (en) | 2021-05-13 | 2021-05-13 | Application of hexahydrobenzophenanthridine alkaloids in protecting dopamine neurons |
| CN202110522481.2A CN113372355B (en) | 2021-05-13 | 2021-05-13 | Hexahydrobenzophenanthridine alkaloid and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110522481.2A CN113372355B (en) | 2021-05-13 | 2021-05-13 | Hexahydrobenzophenanthridine alkaloid and preparation method and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211318400.8A Division CN115518069B (en) | 2021-05-13 | 2021-05-13 | Application of hexahydrobenzophenanthridine alkaloids in protecting dopamine neurons |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113372355A CN113372355A (en) | 2021-09-10 |
| CN113372355B true CN113372355B (en) | 2023-02-17 |
Family
ID=77570877
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110522481.2A Active CN113372355B (en) | 2021-05-13 | 2021-05-13 | Hexahydrobenzophenanthridine alkaloid and preparation method and application thereof |
| CN202211318400.8A Active CN115518069B (en) | 2021-05-13 | 2021-05-13 | Application of hexahydrobenzophenanthridine alkaloids in protecting dopamine neurons |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211318400.8A Active CN115518069B (en) | 2021-05-13 | 2021-05-13 | Application of hexahydrobenzophenanthridine alkaloids in protecting dopamine neurons |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN113372355B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11310530A (en) * | 1998-04-30 | 1999-11-09 | Maruho Co Ltd | Nitrogen monoxide production inhibitor |
| CN1878775A (en) * | 2003-11-05 | 2006-12-13 | 措泽·B.·扎拉马 | Novel chelidonine derivatives, methods for the production thereof, and use thereof for producing pharmaceutical agents |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1254243C (en) * | 2001-05-11 | 2006-05-03 | 殷载淳 | Pharmaceutical compositions comprising chelidonine or derivatives thereof |
| EP1459753A1 (en) * | 2003-03-18 | 2004-09-22 | Nowicky, Wassyl, Dipl.-Ing. DDr. | Quaternary chelidonine and alkaloid derivatives, process for their preparation and their use in the manufacture of medicaments |
| WO2010025272A1 (en) * | 2008-08-27 | 2010-03-04 | The Trustees Of Columbia University In The City Of New York | Compounds, compositions and methods for reducing toxicity and treating or preventing diseases |
| WO2016061509A1 (en) * | 2014-10-17 | 2016-04-21 | The Broad Institute, Inc. | Compositions and methods of treatng muscular dystrophy |
-
2021
- 2021-05-13 CN CN202110522481.2A patent/CN113372355B/en active Active
- 2021-05-13 CN CN202211318400.8A patent/CN115518069B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11310530A (en) * | 1998-04-30 | 1999-11-09 | Maruho Co Ltd | Nitrogen monoxide production inhibitor |
| CN1878775A (en) * | 2003-11-05 | 2006-12-13 | 措泽·B.·扎拉马 | Novel chelidonine derivatives, methods for the production thereof, and use thereof for producing pharmaceutical agents |
Non-Patent Citations (6)
| Title |
|---|
| "1177495-22-5/rn";ACS;《STN REGISTRY》;20090828;1 * |
| "Alkaloids from Dactylicapnos torulosa";Ruecker, G. et al.,;《Phytochemistry》;19941231;第36卷(第2期);519-23 * |
| "Chelidonine inhibits TNF-α-induced inflammation by suppressing the NF-κB pathways in HCT116 cells";Zhang, Zhi Hong et al.,;《Phytotherapy Research 》;20181231;第31卷(第1期);65-75 * |
| "Niosomes of active Fumaria officinalis phytochemicals: antidiabetic, antineuropathic,anti‑inflammatory, and possible mechanisms of action";Karim M. Raafat et al.,;《Chinese Medicine》;20200501;1-22 * |
| "小藤铃儿草的生物碱成分";张国林等;《药学学报》;19901231;第25卷(第8期);604-607 * |
| Zhang, Guo-Lin et al.,."Alkaloids from Dactylicapnos torulosa".《Phytochemistry》.1995,第40卷(第1期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113372355A (en) | 2021-09-10 |
| CN115518069A (en) | 2022-12-27 |
| CN115518069B (en) | 2023-11-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105920064A (en) | Natural active ingredient extracted and separated from leaves and stems of panax quinuefolium L and application of natural active ingredient | |
| CN111228254A (en) | Application and preparation method of phenolic compound ZKYY-041 | |
| CN113372355B (en) | Hexahydrobenzophenanthridine alkaloid and preparation method and application thereof | |
| CN111228246A (en) | Application and preparation method of terpene phenol | |
| CN106939031B (en) | A kind of spirosolane type glycoalkaloid and preparation method thereof and purposes | |
| CN112194698A (en) | Triterpenoid compound and preparation method and application thereof | |
| CN105712844A (en) | Cadinane sesquiterpene Saliciforliusin A and preparation method and application thereof | |
| CN106928309B (en) | A kind of spirosolane type glycoalkaloid and preparation method thereof and purposes | |
| CN110330544A (en) | A kind of bicyclic steroid of 4,4,1- and its preparation method and application | |
| CN105646151B (en) | A kind of purposes of acetylene compound and preparation method thereof and the compound | |
| CN102203099B (en) | Novel compound with spiro chiral carbon backbone, preparation method thereof, and pharmaceutical composition containing same | |
| CN110204589B (en) | Effective component of feather cockscomb seed, extraction method and application thereof in preparing neuroprotective medicament | |
| CN112194697B (en) | Novel triterpenoid and application thereof in preparation of medicine for treating cardiovascular diseases | |
| CN111184710A (en) | Application and preparation method of cyclic phenol | |
| CN108129538B (en) | Method for extracting antibacterial compound from scorpion | |
| CN113480557B (en) | Polyketone compounds, preparation method thereof and application thereof in preparation of antitumor drugs | |
| CN107789429A (en) | The isolation and purification method of bacopa monnieri total saponin extracts and its application in drugs for nervous is prepared | |
| CN108822175B (en) | 3, 16-androstenedione compound and application thereof | |
| CN104398532B (en) | Application of cardiac glycoside compound 12beta-hydroxycalotropin | |
| CN111620819B (en) | Separation and purification method and application of two compounds in musa basjoo root | |
| CN112047887B (en) | Tinospora sinensis amide and preparation method and application thereof | |
| CN117186166B (en) | Aromatic steroid compound, preparation method and application thereof | |
| CN109503623B (en) | Guanactastane compound, preparation method thereof and application thereof in preparation of antibacterial drugs | |
| CN108558975B (en) | 12 beta-hydroxy-androstane 4, 14-diene-16-ketone compound and application thereof | |
| CN106966969B (en) | A kind of alkaloid compound and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |