CN109942592B - Novel protopine type alkaloid and preparation method and application thereof - Google Patents

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

A new proto-opioid alkaloid and its preparation method and application

technical field

The invention belongs to the technical field of phytochemistry, and in particular relates to a new proto-opioid alkaloid and a preparation method and application thereof.

Background technique

Xia Wu, also known as a golden elixir, a fairy in a cave, a drop of water, etc., is the tuber of Corydalis decumbens, a plant belonging to the genus Corydalis decumbens of the Poppy family. Because its ground part withers in early summer and is hard to find, it is named Xia Wu. It is absent in summer, bitter in nature, slightly acrid, and returns to the liver meridian. In summer, it has the functions of activating Qi and relieving pain, promoting blood circulation and removing blood stasis. As a common folk herbal medicine, Xiaxia Wu is often used to treat hypertension, polio sequelae, sciatica, bruises and other diseases. It has pharmacological effects such as dilating blood vessels, directly relaxing smooth muscle, anti-platelet aggregation, and anti-inflammatory. Modern research has found that Xiawuzhong contains a variety of alkaloids, and has a good effect of calming the mind and nourishing intelligence and treating nervous system diseases. However, up to now, there is no report on the related chemical constituents of Xianwu in antidepressant research.

SUMMARY OF THE INVENTION

In view of the above-mentioned situation, the first purpose of the present invention is to provide a new proto-opioid alkaloid, the second purpose is to provide a preparation method of the above-mentioned new proto-opioid alkaloid, and the third purpose is to provide the application of the alkaloid in antidepressant.

In order to achieve the above object, the concrete scheme adopted in the present invention is:

A new proto-opioid alkaloid, its molecular formula is C ₃₁ H ₃₄ N ₂ O ₆ , and its structural formula is

The present invention also provides a method for preparing the above-mentioned neoproto-opioid alkaloids, comprising the following steps:

Step 1. Take the tubers that are not available in summer, pulverize them to 30-50 mesh, place them in an 80-95% ethanol aqueous solution, and soak them at room temperature for 10-15 hours; then heat and reflux for extraction 3 times, each time 4 -6 hours, stand for filtration, combine the filtrates, dry under reduced pressure at low temperature until there is no alcohol smell, and obtain the total extract without summer;

Step 2: Evenly suspend the total extract of Xianwu in step 1 in a hydrochloric acid solution with a volume fraction of 1.0%-2.0%, stand for 25-30 hours, and filter to obtain a supernatant;

Step 3. The supernatant obtained in step 2 is adjusted to alkaline solution I and alkaline solution II with different pHs with ammonia water with a volume concentration of 1.5%-2.0%. The pH of the alkaline solution I is 9.0-10.0, so The pH of the alkaline solution II is 11.0-12.0; the alkaline solution I and the alkaline solution II are combined to obtain a mixed solution; the mixed solution is sequentially extracted with petroleum ether, chloroform and n-butanol to obtain petroleum ether extract, chloroform respectively. Extract and n-butanol extract; Concentrate and dry the petroleum ether extract, chloroform extract and n-butanol extract respectively to obtain extract I, extract II and extract III;

Step 4: Screening the extract I, extract II and extract III obtained in step three by using the reuptake inhibition experiment of monoamine neurotransmitters in vitro, the results show that the extract II has antidepressant activity;

Step 5. Mix the extract II thick paste with antidepressant activity and 100-200 mesh silica gel and load the sample onto the column; use petroleum ether-acetic acid with a volume ratio of 20:1, 12:1, 9:1, 6:1 The ethyl ester elution system performs gradient elution successively to obtain fraction A, fraction B, fraction C and fraction D;

Step 6: Suspend fraction C in petroleum ether, and wet the silica gel column; use petroleum ether-ethyl acetate elution system with volume ratios of 10:1, 8:1, 6:1 to carry out gradient elution successively, Obtain C-1 sub-fraction, C-2 sub-fraction and C-3 sub-fraction respectively;

Step 7. Put the C-3 sub-fraction on a Sephadex LH-20 gel column and elute with 90%-100% methanol to obtain C-3-1 fraction, C-3-2 fraction and C-3- 3 fractions;

Step 8: Prepare a liquid phase on the C-3-2 sub-fraction, carry out separation and purification, and obtain a new compound, that is, a neoproto-opioid alkaloid, after multiple preparations and purifications.

As a further optimization of the above scheme, the mesh number of the silica gel in the silica gel column described in step 6 is 200-300 mesh.

As a further optimization of the above scheme, the parameters for separation and purification in step 8 are: mobile phase 80-100% methanol, absorption wavelength 235 nm, flow rate: 3-6 mL/min.

The present invention further provides the application of the neoproto-opioid alkaloids in the preparation of antidepressant drugs.

Beneficial effects:

The present invention has the advantages that the obtained compound has novel structure and significant antidepressant activity, and the preparation method is simple and easy, the yield is high and the purity is good, which is convenient for further pharmacological and clinical research.

Description of drawings

Fig. 1 is the preparation flow chart of neoproto-opioid alkaloids described in Example 1;

Figure 2 is a coupling correlation diagram of the neoproto-opioid alkaloid compounds of the present invention.

Detailed ways

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 kind of preparation method of neoproto-opioid alkaloid, the concrete steps are as follows:

(1) pulverize 10.0 kg of tubers without summer to 40 mesh, place in 90% ethanol, soak at room temperature for 12 hours; then extract by heating and refluxing 3 times, 5 hours each time, stand to filter, combine filtrate, low temperature It was dried under reduced pressure until there was no alcohol odor, and the total extract (857.5 g) was obtained without summer;

(2) Suspended the Xianwu total extract in 1.5% hydrochloric acid solution, let stand for 28 hours, and filtered to obtain the supernatant;

(3) the obtained supernatant is adjusted to two kinds of alkaline solutions of pH=9 and pH=11 with 1.8% ammonia water respectively, and two kinds of alkaline solutions are combined to obtain a mixed solution; the mixed solution is successively used with petroleum ether, Extracted with chloroform and n-butanol, and then concentrated and dried to obtain petroleum ether fractional extract (38.7 g), chloroform fractional extract (46.2 g), and n-butanol fractional extract (524.6 g);

(4) The reuptake inhibition experiments of monoamine neurotransmitters in vitro were used to screen the extracts of the three parts obtained above (see the activity screening section below for the specific methods), and the screening results showed that the chloroform part extracts had significant antidepressant properties active;

(5) The thick paste of the chloroform fraction extract with antidepressant activity is mixed with silica gel (100-200 mesh) and loaded onto the column. Gradient elution was carried out using a petroleum ether-ethyl acetate elution system with a volume ratio of 20:1→12:1→9:1→6:1, respectively, to obtain fraction A (6.6 g) and fraction B (11.7 g) , C fraction (15.3 g), D fraction (8.7 g) 4 fractions;

(6) Suspend the C fraction in petroleum ether, and wet it onto a silica gel (200-300 mesh) column. Gradient elution was carried out sequentially with petroleum ether-ethyl acetate with a volume ratio of 10:1→8:1→6:1 to obtain C-1 sub-fraction (3.1 g) and C-2 sub-fraction (6.2 g) 3 sub-fractions including C-3 sub-fraction (5.2 g);

(7) The C-3 sub-fraction was dissolved in methanol, applied to a Sephadex LH-20 gel column, and eluted with 90%, 95%, and 100% methanol in turn to obtain the C-3-1 sub-fraction (1.1 g), C-3-2 sub-fraction (2.2 g), C-3-3 sub-fraction (1.4 g) and other 3 sub-fractions;

(8) Prepare the liquid phase on the C-3-2 second fraction, and carry out separation and purification (mobile phase: 85% methanol, absorption wavelength: 235 nm, flow rate: 5 mL/min), and finally obtain a new compound after multiple preparations and purifications (10.25 mg).

The compound obtained in the present invention is a colorless powder, HR-ESI-MS m/z 553.2018 [M+Na] ⁺ (calcd. for C ₃₁ H ₃₄ N ₂ O ₆ Na, 553.2162). UV(MeOH)λ _max : 210, 235, 322 nm; IR v _max : 2903, 1712, 1622, 1462, 1386 cm ⁻¹ ; ¹ H NMR (CDCl ₃ , 400 MHz) and ¹³ C NMR (CDCl ₃ , 100 MHz) data are shown in the table 1. The structure of the compound was identified using its spectral data, references and its coupling-related information such as HMBC, 2D-NOESY, ¹ H- ¹ H COSY (Fig. 2). After SciFinder search, it was found that the compound was a neoproto-opioid alkaloid.

Table 1: ¹ H NMR, ¹³ C NMR and HMBC related data for compounds

Test example

The antidepressant activity screening of the above-mentioned new compounds is as follows:

1. Experimental method

1.1. Animals and Medicines

Male SD rats, body weight: 200-230 g. Breeding room level; SPF animal room is routinely raised. 6-Hydroxydopamine, desipramine and fluoxetine hydrochloride were purchased from Sigma.

1.2. Test sample preparation

The new compound was dissolved in 10% DMSO solvent to make a final concentration of 100 μg/ml.

1.3. Preparation of reference substance

Dissolve 6-hydroxydopamine (1.03mg) in 0.5ml distilled water, the final reaction concentration is 0.1mM; Dissolve desipramine (1.51mg) in 0.5ml distilled water, the final reaction concentration is 0.1mM; Ting (1.73 mg) was dissolved in 0.5 ml of distilled water, and the final reaction concentration was 0.1 mM.

1.4. Preparation of brain synaptosomes

After the rat was decapitated, the brain was quickly taken out and placed in pre-cooled (4°C) normal saline to remove the pia mater and vascular tissue. Take 3 g of cerebral cortex and put it into 30 ml (0.32 M) of cold sugar solution. Homogenize with an ultrasonic cell crusher, equilibrate centrifugation (1500g, 10min) at 4°C, then take the supernatant and centrifuge (20000g, 30min), discard the supernatant, and keep the precipitate, that is, the crude extract of synaptosomes. The precipitated part was then suspended with 0.32M cold sucrose solution and carefully spread on the 1.2M and 0.8M (10ml each) cold sucrose gradient solution from the bottom of the tube, and centrifuged at 4°C for equilibration (38000g, 60min). Carefully collect the suspended band at the interface of 0.8-1.2M sucrose with a puncture needle, place it in 10ml (0.32M) cold sucrose solution and mix, and then equilibrate and centrifuge again at 4°C (20000g, 30min), and the precipitate is the refined brain. synaptosome. The precipitate was suspended in a small amount of Tris-Krebs buffer, and the protein content in the suspension was measured with a total protein assay reagent.

1.5. Reuptake inhibition experiments of monoamine neurotransmitters

First add 1.0ml Tris-Krebs buffer to the test tube, then add 20μl synaptosome suspension, then add 10μl control sample or drug to be tested, mix well, and warm in a 37°C water bath for 5min. Add 10 μl of substrates ( ³ H-5HT, ³ H-NA, ³ H-DA; final reaction concentration: 300 nM) in a 4°C environment, mix well, and incubate in a 37°C water bath for 5 min. Subsequently, 3 ml of cold Tris-Krebs buffer was added to each tube to stop the reaction, and the cells were quickly filtered through a glass fiber filter with a multi-head cell harvester, and the test tube and filter were washed twice with the same volume of the same solution. Remove the filter, dry at 60-70°C, put the filter into a scintillation vial, add toluene scintillation fluid, and count with a β-liquid scintillation counter. The final reaction concentrations of the positive control drugs (6-hydroxydopamine, desipramine, and fluoxetine hydrochloride) were all 0.1 mM. At this concentration, the positive control drug can 100% inhibit the reuptake of 5-HT, NA and DA by rat brain synaptosomes. Therefore, taking 1 mM as the final reaction concentration of the positive control drug, the inhibitory effect of the sample to be tested is compared with the corresponding positive control drug to screen for the inhibitory effect.

2. Experimental results

In this experiment, 6-hydroxydopamine, desipramine, and fluoxetine hydrochloride were assumed to inhibit the reuptake of ³ H-5HT, ³ H-NA, and ³ H-DA by 100%. From the results of the reuptake inhibition experiment of monoamine neurotransmitters in vitro (Table 2), it can be seen that the new compound can significantly inhibit the reuptake of 5-HT, which is comparable to the positive control drug fluoxetine hydrochloride; the new compound can inhibit the reuptake of NE. Uptake has a certain degree of inhibitory effect, but its inhibitory rate is slightly weaker than that of the positive control drug desipramine; the new compound has a certain degree of inhibitory effect on the monoamine neurotransmitter DA, but its inhibitory rate is weaker than that of the positive control drug6 -Hydroxydopamine. The above studies found that the neoproto-opioid alkaloids isolated from Xianwu have important antidepressant activities, which lays an important theoretical and experimental basis for the discovery of new antidepressant drugs from Xianwu in the future.

Table 2: Inhibitory effects of new compounds on the uptake of 5-HT, NE and DA in rat brain synaptosomes

The present invention selects Jiangxi Daodi medicinal material, Xianwu, as the research object, which is very innovative. Through the research, we find that the chloroform extracted part of Xiawu has good antidepressant activity. Therefore, the present invention uses modern extraction and separation technology to continue to carry out in-depth research on the active part of Xianwu, and isolates a new proto-opioid alkaloid from it. At the same time, it is found that the new compound has significant antidepressant activity. It has laid an important theoretical and experimental basis for the development of antidepressant drugs with independent intellectual property rights.

It should be noted that the above-mentioned embodiments should be construed as illustrative rather than limiting the protection scope of the present invention, which is subject to the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, some non-essential improvements and adjustments made to the present invention still belong to the protection scope of the present 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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