CN109942592B - Novel protopine type alkaloid and preparation method and application thereof - Google Patents
Novel protopine type alkaloid and preparation method and application thereof Download PDFInfo
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- GPTFURBXHJWNHR-UHFFFAOYSA-N protopine Chemical compound C1=C2C(=O)CC3=CC=C4OCOC4=C3CN(C)CCC2=CC2=C1OCO2 GPTFURBXHJWNHR-UHFFFAOYSA-N 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 229930013930 alkaloid Natural products 0.000 title claims description 10
- 150000003797 alkaloid derivatives Chemical class 0.000 title claims description 7
- GTRPODKMSBFDOI-UHFFFAOYSA-N Protopine Natural products CN1Cc2c3OCOc3ccc2C4C1Cc5cc6OCOc6cc5C4=O GTRPODKMSBFDOI-UHFFFAOYSA-N 0.000 title claims description 4
- ZAALQOFZFANFTF-UHFFFAOYSA-N Pseudoprotipine Natural products C1=C2C(=O)CC3=CC=4OCOC=4C=C3CN(C)CCC2=CC2=C1OCO2 ZAALQOFZFANFTF-UHFFFAOYSA-N 0.000 title claims description 4
- CHWPMFMUQATVNK-ARYYTZDLSA-N dihydrosporogen AO-1 Natural products O[C@H]1[C@]2(C(C)=C)O[C@@H]2[C@]2(C)[C@@H](C)[C@H](O)CCC2=C1 CHWPMFMUQATVNK-ARYYTZDLSA-N 0.000 title claims description 4
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- 229940005513 antidepressants Drugs 0.000 claims abstract description 18
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- GIYXAJPCNFJEHY-UHFFFAOYSA-N N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-1-propanamine hydrochloride (1:1) Chemical compound Cl.C=1C=CC=CC=1C(CCNC)OC1=CC=C(C(F)(F)F)C=C1 GIYXAJPCNFJEHY-UHFFFAOYSA-N 0.000 description 5
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- DIVDFFZHCJEHGG-UHFFFAOYSA-N oxidopamine Chemical compound NCCC1=CC(O)=C(O)C=C1O DIVDFFZHCJEHGG-UHFFFAOYSA-N 0.000 description 5
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- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a novel protopine alkaloid, a preparation method and application thereof, and belongs to the technical field of phytochemistryC31H34N2O6The novel compound is separated and extracted from the active site of corydalis amabilis, and the novel protopine alkaloid of the novel compound is found to have obvious antidepressant activity, thereby laying important theoretical basis and experimental basis for developing antidepressant medicaments with independent intellectual property rights from corydalis amabilis in China.
Description
Technical Field
The invention belongs to the technical field of phytochemistry, and particularly relates to a novel protopine alkaloid, and a preparation method and application thereof.
Background
Rhizoma Corydalis Decumbentis, also called YIJINDAN, DONGLIANSHENXIAN, and SHUIZHU, is tuber of Corydalis Tuberosa (Corydalis Decumbens) belonging to Corydalis of Papaveraceae. Its overground part withers and is hard to find its trail in the early summer, so that it is named as corydalis amabilis. Corydalis amabilis, bitter in nature, slightly pungent and entering liver meridian. Rhizoma corydalis Decumbentis has effects of activating qi-flowing, relieving pain, promoting blood circulation, and removing blood stasis. Rhizoma corydalis Decumbentis is a folk common herbal medicine, and is used for treating hypertension, poliomyelitis sequela, sciatica, traumatic injury, etc. It has pharmacological effects of dilating blood vessel, directly relaxing smooth muscle, resisting platelet aggregation, and resisting inflammation. Modern researches find that rhizoma corydalis Decumbentis contains various alkaloids, and has good effects of calming heart, improving intelligence and treating nervous system diseases. However, no relevant chemical components are reported in the research on the antidepressant aspect of corydalis amabilis up to now.
Disclosure of Invention
In view of the above circumstances, the present invention provides a novel protopine alkaloid, a second object is to provide a method for preparing the novel protopine alkaloid, and a third object is to provide an application of the alkaloid in antidepressant.
In order to achieve the purpose, the invention adopts the specific scheme that:
a novel protopine type alkaloid with molecular formula of C31H34N2O6Structural formula is
The invention also provides a preparation method of the novel protopine alkaloid, which comprises the following steps:
firstly, tubers of corydalis amabilis are taken and crushed into 30-50 meshes, and are placed in 80-95% ethanol water solution by volume fraction to be soaked for 10-15 hours at room temperature; then heating and reflux extracting for 3 times, each time for 4-6 hr, standing and filtering, mixing filtrates, drying at low temperature under reduced pressure until no alcohol smell exists to obtain rhizoma corydalis Decumbentis total extract;
step two, uniformly suspending the total corydalis amabilis extract obtained in the step one in a hydrochloric acid solution with the volume fraction of 1.0-2.0%, standing for 25-30 hours, and filtering to obtain a supernatant;
step three, respectively adjusting the supernatant obtained in the step two into alkaline liquid I and alkaline liquid II with different pH values by using ammonia water with the volume concentration of 1.5-2.0%, wherein the pH value of the alkaline liquid I is 9.0-10.0, and the pH value of the alkaline liquid II is 11.0-12.0; combining the alkaline solution I and the alkaline solution II to obtain a mixed solution; sequentially extracting the mixed solution with petroleum ether, chloroform and n-butanol to respectively obtain petroleum ether extract, chloroform extract and n-butanol extract; concentrating and drying the petroleum ether extract, the chloroform extract and the n-butanol extract respectively to obtain an extract I, an extract II and an extract III;
step four, screening the extract I, the extract II and the extract III obtained in the step three by adopting a reuptake inhibition experiment of in vitro monoamine neurotransmitters, wherein the result shows that the extract II has antidepressant activity;
step five, mixing the extract II thick paste with the antidepressant activity and 100 and 200 meshes of silica gel, and loading the mixture on a column; sequentially carrying out gradient elution by adopting petroleum ether-ethyl acetate elution systems with volume ratios of 20:1, 12:1, 9:1 and 6:1 to respectively obtain fraction A, fraction B, fraction C and fraction D;
suspending the fraction C in petroleum ether, and applying a silica gel column by a wet method; sequentially carrying out gradient elution by adopting petroleum ether-ethyl acetate elution systems with volume ratios of 10:1, 8:1 and 6:1 to respectively obtain C-1 sub-fraction, C-2 sub-fraction and C-3 sub-fraction;
seventhly, putting the C-3 sub-fraction on a Sephadex L H-20 gel column, and eluting with 90-100% methanol to obtain C-3-1 fraction, C-3-2 fraction and C-3-3 fraction;
and step eight, preparing a liquid phase on the C-3-2 fractions, separating and purifying, and preparing and purifying for multiple times to obtain a new compound, namely the new protopine alkaloid.
As a further optimization of the scheme, the mesh number of the silica gel in the silica gel column in the step six is 200-300 meshes.
As a further optimization of the scheme, the parameters of the separation and purification in the step eight are that the mobile phase contains 80-100% of methanol, the absorption wavelength is 235nm, and the flow rate is 3-6m L/min.
The invention also provides application of the novel protopine alkaloid in the aspect of preparing antidepressant medicaments.
Has the advantages that:
the invention has the advantages that the obtained compound has novel structure, obvious antidepressant activity, simple and easy preparation method, high yield and good purity, and is convenient for further pharmacological and clinical research.
Drawings
FIG. 1 is a flow chart of the preparation of the novel protopine-type alkaloids described in example 1;
FIG. 2 is a graph relating to the coupling of the novel protopine-type alkaloid compounds of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1:
a method for preparing protopine alkaloid comprises the following steps:
(1) crushing 10.0 kg of corydalis tuber to 40 meshes, putting into 90% ethanol, and soaking for 12 hours at room temperature; then heating and refluxing for extraction for 3 times, each time for 5 hours, standing and filtering, combining the filtrates, drying at low temperature under reduced pressure until no alcohol smell exists to obtain rhizoma corydalis Decumbentis total extract (857.5 g);
(2) suspending rhizoma corydalis Decumbentis total extract in 1.5% hydrochloric acid solution, standing for 28 hr, and filtering to obtain supernatant;
(3) adjusting the obtained supernatant to two alkaline solutions with pH 9 and pH 11 respectively with 1.8% ammonia water, and mixing the two alkaline solutions to obtain a mixed solution; sequentially extracting the mixed solution with petroleum ether, chloroform and n-butanol, and concentrating and drying to respectively obtain petroleum ether extract (38.7 g), chloroform extract (46.2 g) and n-butanol extract (524.6 g);
(4) screening the obtained 3 part extracts respectively by adopting an in-vitro monoamine neurotransmitter re-uptake inhibition experiment (the specific method is shown in the following activity screening part), and the screening result shows that the chloroform part extract has remarkable antidepressant activity;
(5) mixing the thick paste of chloroform fraction extract with silica gel (100 and 200 mesh) and loading on column. Gradient elution is carried out by adopting a petroleum ether-ethyl acetate elution system with the volume ratio of 20:1 → 12:1 → 9:1 → 6:1 respectively to obtain 4 fractions of fraction A (6.6 g), fraction B (11.7 g), fraction C (15.3 g) and fraction D (8.7 g);
(6) the C fraction was suspended in petroleum ether and wet-packed on a silica gel (200-300 mesh) column. Carrying out gradient elution by adopting petroleum ether-ethyl acetate with the volume ratio of 10:1 → 8:1 → 6:1 respectively to obtain 3 sub-fractions such as C-1 sub-fraction (3.1 g), C-2 sub-fraction (6.2 g), C-3 sub-fraction (5.2 g) and the like;
(7) dissolving the C-3 sub-fraction with methanol, loading on Sephadex L H-20 gel column, and sequentially eluting with 90%, 95%, and 100% methanol to obtain 3 sub-fractions such as C-3-1 sub-fraction (1.1 g), C-3-2 sub-fraction (2.2 g), and C-3-3 sub-fraction (1.4 g);
(8) the liquid phase was prepared from the C-3-2 cut fraction, and separation and purification (mobile phase: 85% methanol, absorption wavelength: 235nm, flow rate: 5m L/min) were carried out to obtain a novel compound (10.25 mg) through multiple preparations and purifications.
The compound obtained by the invention is colorless powder, HR-ESI-MS M/z 553.2018[ M + Na [ ]]+(calcd.forC31H34N2O6Na，553.2162)。UV(MeOH)λmax:210，235，322nm；IRνmax:2903，1712，1622，1462，1386cm-1；1H NMR(CDCl3400MHz) and13C NMR(CDCl3100MHz) data are shown in Table 1. The spectral data of the compounds, the references and their HMBC, 2D-NOESY,1H-1H COSY and other coupling related information (figure 2) identifies the structure of the protein. The SciFinder search finds that the compound is a novel protopine alkaloid.
Table 1: process for preparing compounds1H NMR、13C NMR and HMBC related data
Test examples
The screening of the antidepressant activity of the new compound is as follows:
1. experimental methods
1.1 animals and drugs
Male SD rats, body weight: 200- & gt 230 g. Feeding house level; SPF-level animal houses are conventionally raised. 6-hydroxydopamine, desipramine and fluoxetine hydrochloride were purchased from Sigma.
1.2 test sample preparation
The novel compounds were dissolved in 10% DMSO solvent to prepare a solution having a final concentration of 100. mu.g/ml.
1.3 preparation of control
6-hydroxydopamine (1.03mg) was dissolved in 0.5ml of distilled water to a final reaction concentration of 0.1 mM; decipipramine (1.51mg) was dissolved in 0.5ml of distilled water to give a final reaction concentration of 0.1 mM; fluoxetine hydrochloride (1.73mg) was dissolved in 0.5ml of distilled water to give a final reaction concentration of 0.1 mM.
1.4 preparation of brain synaptosome
After decapitation, the brain of the rat was quickly removed and placed in pre-cooled (4 ℃) physiological saline to remove the pia mater and vascular tissue. 3g of cerebral cortex was taken and put into 30ml (0.32M) of a cooled sucrose solution. Homogenizing with ultrasonic cell disruptor, equilibrating and centrifuging at 4 deg.C (1500g, 10min), then centrifuging the supernatant (20000g, 30min), discarding the supernatant, and retaining the precipitate, i.e., the crude extract of synaptosomes. The pellet was suspended in 0.32M cold sucrose solution and carefully applied to 1.2M and 0.8M (10 ml each) cold sucrose gradient from the bottom of the tube, followed by equilibration centrifugation at 4 deg.C (38000g, 60 min). Carefully collecting the suspension band at the sucrose interface of 0.8-1.2M with a puncture needle, mixing in 10ml (0.32M) of cold sucrose solution, and performing equilibrium centrifugation at 4 deg.C (20000g, 30min) to obtain the precipitate as refined brain synaptosome. The precipitate is suspended in a small amount of Tris-Krebs buffer solution, and the total protein determination reagent is used for measuring the protein content in the suspension.
1.5 reuptake inhibition assay for monoamine neurotransmitters
1.0ml of Tris-Krebs buffer solution is added into the test tube, 20 mu l of synaptosomal suspension is added, 10 mu l of control sample or drug to be detected is added, the mixture is mixed evenly, and the mixture is bathed in water bath at 37 ℃ for 5 min. Adding 10 μ l of substrate(s) at 4 ℃3H-5HT、3H-NA、3H-DA and final reaction concentration of 300nM), uniformly mixing, carrying out warm bath in a water bath at 37 ℃ for 5min, adding 3ml of cold Tris-Krebs buffer solution into each tube to terminate the reaction, rapidly carrying out suction filtration by a multi-head cell collector through a glass fiber filter membrane, washing the test tube and the filter for 2 times by using the same solution with the same volume, taking down the filter membrane, drying at 60-70 ℃, putting the filter membrane into a scintillation bottle, adding toluene scintillation liquid, counting by using an β -liquid flash counter, wherein the final reaction concentrations of positive control drugs (6-hydroxydopamine, desipramine and fluoxetine hydrochloride) are all 0.1 mM., and the positive control drugs can inhibit the reuptake of 5-HT, NA and DA by 100% of rat brain synaptosome, so that 1mM is used as the final reaction concentration of the positive control drugs, and the inhibition of a sample to be detected is compared with the corresponding positive control drugs to carry out inhibition screening.
2. Results of the experiment
The experiment shows that 6-hydroxydopamine, desipramine and fluoxetine hydrochloride can inhibit 100 percent3H-5HT、3H-NA、3Reuptake of H-DA. From in vitro monoamine neurotransmittersThe results of the reuptake inhibition experiment (table 2) show that the new compound can obviously inhibit the reuptake of 5-HT, and is equivalent to the positive control drug fluoxetine hydrochloride; the new compound has a certain degree of inhibition effect on the reuptake of NE, but the inhibition rate of the new compound is slightly weaker than that of a positive control drug of desipramine; the novel compound has a certain degree of inhibition effect on monoamine neurotransmitter DA, but the inhibition rate of the novel compound is weaker than that of a positive control drug 6-hydroxydopamine. The research discovers that the novel protopine alkaloid separated from the corydalis amabilis has important antidepressant activity, and lays an important theoretical basis and experimental basis for finding novel antidepressant drugs from the corydalis amabilis in future.
Table 2: inhibition of 5-HT, NE, DA uptake by rat brain synaptosomes
The invention selects medicinal materials in the way of Jiangxi-rhizoma corydalis Decumbentis as research objects to research the antidepressant activity, and researches show that chloroform extraction parts of rhizoma corydalis Decumbentis have good antidepressant activity. Therefore, the invention utilizes the modern extraction and separation technology to continuously carry out deep research on the active site of the corydalis amabilis, separates and obtains a new protopine alkaloid, and simultaneously discovers that the new compound has obvious antidepressant activity, thereby laying important theoretical basis and experimental basis for developing antidepressant medicaments with independent intellectual property rights from the corydalis amabilis in China.
It is to be noted, however, that the above-mentioned embodiments are to be considered illustrative rather than limiting of the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
Claims (5)
1. A novel protopine type alkaloid with molecular formula of C31H34N2O6Structural formula is
2. The process for the preparation of a novel protopine-type alkaloid according to claim 1, wherein: the method comprises the following steps:
firstly, tubers of corydalis amabilis are taken and crushed into 30-50 meshes, and are placed in 80-95% ethanol water solution by volume fraction to be soaked for 10-15 hours at room temperature; then heating and reflux extracting for 3 times, each time for 4-6 hr, standing and filtering, mixing filtrates, drying at low temperature under reduced pressure until no alcohol smell exists to obtain rhizoma corydalis Decumbentis total extract;
step two, uniformly suspending the total corydalis amabilis extract obtained in the step one in a hydrochloric acid solution with the volume fraction of 1.0-2.0%, standing for 25-30 hours, and filtering to obtain a supernatant;
step three, respectively adjusting the supernatant obtained in the step two into alkaline liquid I and alkaline liquid II with different pH values by using ammonia water with the volume concentration of 1.5-2.0%, wherein the pH value of the alkaline liquid I is =9.0-10.0, and the pH value of the alkaline liquid II is = 11.0-12.0; combining the alkaline solution I and the alkaline solution II to obtain a mixed solution; sequentially extracting the mixed solution with petroleum ether, chloroform and n-butanol to respectively obtain petroleum ether extract, chloroform extract and n-butanol extract; concentrating and drying the petroleum ether extract, the chloroform extract and the n-butanol extract respectively to obtain an extract I, an extract II and an extract III;
step four, screening the extract I, the extract II and the extract III obtained in the step three by adopting a reuptake inhibition experiment of in vitro monoamine neurotransmitters, wherein the result shows that the extract II has antidepressant activity;
step five, mixing the extract II thick paste with the antidepressant activity and 100 and 200 meshes of silica gel, and loading the mixture on a column; sequentially carrying out gradient elution by adopting petroleum ether-ethyl acetate elution systems with volume ratios of 20:1, 12:1, 9:1 and 6:1 to respectively obtain fraction A, fraction B, fraction C and fraction D;
suspending the fraction C in petroleum ether, and applying a silica gel column by a wet method; sequentially carrying out gradient elution by adopting petroleum ether-ethyl acetate elution systems with volume ratios of 10:1, 8:1 and 6:1 to respectively obtain C-1 sub-fraction, C-2 sub-fraction and C-3 sub-fraction;
seventhly, putting the C-3 sub-fraction on a Sephadex L H-20 gel column, and eluting with 90-100% methanol to obtain C-3-1 fraction, C-3-2 fraction and C-3-3 fraction;
and step eight, preparing a liquid phase from the C-3-2 fractions, separating and purifying, and preparing the liquid phase and purifying for multiple times to obtain a new compound, namely the new protopine alkaloid.
3. The method of claim 2, wherein: the mesh number of the silica gel in the silica gel column in the step six is 200-300 meshes.
4. The preparation method according to claim 2, wherein the separation and purification parameters in step eight are 80-100% methanol as a mobile phase, 235nm absorption wavelength, and 3-6m L/min flow rate.
5. Use of a novel protopine-type alkaloid according to claim 1 in the preparation of an antidepressant.
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