CN110279738A - A kind of extracting method of antidepressant spermidine active component and the purposes of spermidine effective part extract - Google Patents

Abstract

The invention belongs to the technical field of extraction of effective parts of traditional Chinese medicine, and provides a method for extracting antidepressant effective parts of spermidine and the application of the extract of effective parts of spermidine. Dried safflower or safflower dregs, reflux extraction with 60-90% ethanol, concentrated under reduced pressure, the concentrated solution was subjected to medium-pressure rapid purification on a C18 medium-pressure preparative column, eluted with 10-100% ethanol or water, concentrated and dried to obtain sub Spermine effective fraction extract. The effective part of spermidine can significantly reduce the immobility time in the mouse forced swimming test and the mouse tail suspension test, and the effective part of spermidine has obvious antidepressant effect. The active part of spermidine provided by the present invention is a natural antidepressant effective substance group with simple preparation process, significant effect, safety and reliability, and easy industrial production, and can be prepared into capsules, tablets, granules, gels, buffers, etc. Dosage forms such as release agent, oral liquid or drop pill are used for antidepressant drugs or health food.

Description

A method for extracting antidepressant effective parts of spermidine and effective parts of spermidine Uses of the extract

technical field

The invention belongs to the technical field of extracting effective parts of traditional Chinese medicines, and in particular relates to a method for extracting effective parts of spermidine which is antidepressant and the use of extracts from effective parts of spermidine.

Background technique

Depression is a common emotional mental illness, mainly manifested as significant and persistent low mood. Patients with mild depression manifest as a bad mood and loss of interest. In severe cases, they manifest as pessimism and despair. , often suicidal tendencies. Depression has the characteristics of high incidence rate, high relapse rate and high disability rate. The World Health Organization has listed it as one of the top ten serious diseases. With the development of society and economy, the pace of life has accelerated, people's pressure has increased, and various emotional impacts have increased, resulting in an increase in the incidence of depression year by year. The serious harm of depression urgently requires researchers to accelerate research and develop antidepressant drugs with better efficacy and higher safety.

There are many treatment methods for depression, such as psychotherapy, sleep deprivation therapy, phototherapy and electroconvulsive therapy, but drug therapy is still the main method, supplemented by psychotherapy. At present, the drugs used to treat depression are mainly chemical drugs, such as tricyclics (imipramine, amitriptyline, doxepin, trimethylimipramine, clomipramine, etc.), monoamine oxidase inhibitors, etc. (Phenelzine, isofazilazine, cypromine, moclobemide, etc. are commonly used in clinical practice), selective 5-HT reuptake inhibitors (fluoxetine, paroxetine, sertraline, siptylan, etc. are commonly used in clinical practice) However, these chemical drugs have problems such as narrow therapeutic spectrum, poor tolerance, large toxic and side effects, and high price. Therefore, discovering and creating drugs with good antidepressant effects from natural products with long-lasting effects and low incidence of adverse reactions has become a hot research topic at home and abroad.

At present, more than 250 chemical components have been isolated from safflower, including flavonoids, alkaloids, polyacetylenes, alkyl glycols, organic acids, steroids, spermidine, etc. Among them, spermidine derivatives are polyamine compounds with (H ₂ N(CH ₂ ) ₃ NH(CH ₂ ) ₄ NH ₂ ) structural units, which are a kind of special components in safflower. It is reported in the literature that they show It has a wide range of biological activities, such as anti-HIV virus, anti-tumor, anti-oxidation, etc., but the role of spermidine in safflower in nervous and mental diseases, especially depression, has not been reported in the literature, and a class of safflower The preparation of spermidine has not been reported yet.

Contents of the invention

The purpose of the present invention is to provide a method for extracting the antidepressant effective part of spermidine and the application of the extract of the effective part of spermidine.

In order to achieve the above object, the present invention provides the following technical scheme: a method for extracting the effective part of antidepressant spermidine, the specific steps are as follows: take dried safflower or safflower dregs, and reflux 60-90% ethanol to extract 1-2 time, concentrated under reduced pressure, the concentrated solution is subjected to medium-pressure rapid purification through a C18 medium-pressure preparative column, W1/W2 is 1-10, the W1 is the quality of the sample to be separated in mg, and W2 is the quality of the C18 column separation material in g Calculated; 10-100% ethanol or water elution, concentration and drying to obtain the effective fraction extract of spermidine.

The obtained spermidine active part extract contains four different configurations of tricoumarinyl-substituted spermidine substances, and the total content is more than 50%.

The application of the extract of effective part of spermidine, the application of the extract of effective part of spermidine in the preparation of antidepressant medicine or health food.

The antidepressant medicine is prepared by adding the extract of effective parts of spermidine and pharmaceutically acceptable auxiliary materials into a pharmaceutically acceptable conventional dosage form.

The dosage form is capsule, tablet, powder, granule, gel, sustained release, oral liquid or drop pill.

The extract of the active part of spermidine contains four different configurations of tricoumaryl-substituted spermidine substances (see Figure 1 for the structure), with a total content of more than 50%, see Figure 2 for details.

The present invention has the following obvious advantages:

The present invention further optimizes the process used as a preparation process for the effective part of spermidine, and meanwhile has important significance for the further recycling of traditional Chinese medicine dregs.

The present invention discovers the medicinal function of the effective part of spermidine, uses it for antidepressant effect, and can be prepared as a medicine for preventing and/or treating depression, so as to be the clinical application of the medicinal or health care function of the effective part of spermidine. Applications break new ground.

The results of classic antidepressant animal model experiments show that the effective fraction of spermidine has obvious antidepressant effect.

The effective part of the spermidine of the present invention has strong pharmacological action, obvious efficacy for preventing, regulating and treating depression, quick onset, small adverse reaction, low price, and good application prospect.

The present invention can use the active part of spermidine alone to prepare medicine or health food for preventing and treating depression.

Description of drawings

Fig. 1 is the chemical structural formula of 4 spermidines;

Figure 2 is the HPLC spectrum of spermidine compounds in safflower dregs (λ=300 nm).

Detailed ways

The present invention is further described in conjunction with specific embodiments. However, these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

For the experimental methods that do not specify specific experimental conditions in the examples, they are usually operated under conventional conditions, or according to the conditions recommended by the manufacturer.

Example 1: A method for preparing the effective part of spermidine with antidepressant effect, comprising the following steps: take dried safflower dregs, reflux extraction with 60% ethanol once, concentrate under reduced pressure, and prepare the concentrated solution through C18 medium pressure The column is prepared by medium-pressure rapid purification, W1/W2 is 1 (mg/g), eluted with 60% ethanol, concentrated and dried to obtain the effective part of spermidine, and the yield can reach 95.79%.

After analysis: the extract of the active part of spermidine contains four different configurations of tricoumaryl-substituted spermidine substances. The specific structure is shown in Figure 1, and the total content is more than 50%. See Figure 2 for details.

Example 2: A method for preparing the effective part of spermidine with antidepressant effect, comprising the following steps: take dried safflower dregs, reflux extraction with 75% ethanol once, concentrate under reduced pressure, and prepare the concentrated solution through C18 medium pressure The column was prepared by medium-pressure rapid purification, W1/W2 was 5 (mg/g), eluted with 50% ethanol, concentrated and dried to obtain the effective fraction of spermidine, and the yield could reach 97.22%.

Example 3: A method for preparing the effective part of spermidine with antidepressant effect, comprising the following steps: take dried safflower dregs, extract once with 90% ethanol under reflux, concentrate under reduced pressure, and prepare the concentrated solution through C18 medium pressure The column was prepared by medium-pressure rapid purification, W1/W2 was 10 (mg/g), eluted with 40% ethanol, concentrated and dried to obtain the effective fraction of spermidine, and the yield could reach 92.79%.

Embodiment 4: a kind of preparation method of the active part of spermidine with antidepressant effect, comprises the following steps: get dry safflower dregs, 75% ethanol reflux extracts 2 times, concentrate under reduced pressure, concentrated solution is prepared through C18 medium pressure The column was prepared by medium-pressure rapid purification, W1/W2 was 5 (mg/g), 50% ethanol was used as the eluting solvent, concentrated and dried to obtain the effective fraction of spermidine, and the yield could reach 93.52%.

Example 5: A method for preparing the effective part of spermidine with antidepressant effect, comprising the following steps: take dried safflower, extract once with 75% ethanol under reflux, concentrate under reduced pressure, and carry out the concentrated solution through a C18 medium pressure preparation column It is prepared by medium-pressure rapid purification, W1/W2 is 5 (mg/g), eluted with 50% ethanol, concentrated and dried to obtain the effective part of spermidine, and the yield can reach 90.32%.

Embodiment 6: Mouse forced swimming test

Experimental instruments and reagents: animal behavior video analysis system (Chengdu Taimeng Technology Co., Ltd.), thermometer, timer, plexiglass cylinder, animal gavage needle. Total spermidine active fraction extract, sodium carboxymethylcellulose (CMC-Na, Tianjin Kaitong Chemical Reagent Co., Ltd.), venlafaxine hydrochloride (Chengdu Kanghong Pharmaceutical Group Co., Ltd.).

Experimental animals and drug administration: Male ICR mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2016-0006), 18-22g, after adaptive feeding for 1 week, were randomly divided into 5 Group, 12 in each group. The dosage of venlafaxine was 0.05g/kg, the dosage of total spermidine effective part extract was 0.1g/kg, 0.05g/kg, 0.025g/kg, and the blank control group was given an equal volume of distilled water. Animals in each group were given intragastric administration once a day according to body weight, 1ml/20g each time, for 14 consecutive days.

Experimental method: 1 hour after the last administration, the mice were placed in a plexiglass tank with a height of 25cm, a diameter of 10cm, and a water depth of 15cm. The water temperature was 23±2°C. Use the animal behavior video analysis system to record the swimming behavior of the mice within 6 minutes, and the immobility time of the mice forced to swim within 4 minutes after analysis (meaning that the mice stop struggling or show a floating state in the water, and only have small limb movements to keep their heads. floating on the water).

Experimental results: Before the experiment and after the experiment, the mice in each group were weighed, and the effect of spermidine on the mouse weight was observed. The results are shown in Table 1, and the experimental data are represented by Mean±SD. It can be seen from Table 1 that there was no significant difference in the weight change of the mice in each group before and after the experiment, indicating that the drug itself did not affect the weight of the animals, and there was no obvious adverse reaction.

Table 1 Effect of spermidine on body weight of mice before and after experiment (g)

The effect of spermidine on the immobility time in the forced swimming test of mice is shown in Table 2, and the data are represented by Mean ± SD. It can be seen from Table 2 that after 14 days of intragastric administration, the immobility time of each administration group was reduced, and compared with the blank control group, except for the high-dose spermidine group, there was a significant difference ( P<0.05 or P <0.01 ).

Table 2 Effect of spermidine on immobility time in forced swimming test of mice (n=12)

Compared with blank control group: * P<0.05 has significant difference, ** P<0.01 has extremely significant difference. Embodiment 7 mouse tail suspension test

Experimental instruments and reagents: animal behavior video analysis system (Chengdu Taimeng Technology Co., Ltd.), thermometer, timer, plexiglass cylinder, animal gavage needle. Total spermidine active fraction extract, sodium carboxymethylcellulose (CMC-Na, Tianjin Kaitong Chemical Reagent Co., Ltd.), venlafaxine hydrochloride (Chengdu Kanghong Pharmaceutical Group Co., Ltd.).

Experimental animals and administration: Experimental animals and administration are the same as in Example 6.

Experimental method: 1 hour after the last administration, stick the tail tip of the mouse on the hanging hook at 1 cm, and hang the mouse into the observation box of the animal behavior video analysis system, and record the desperate behavior of the mouse within 6 minutes. The immobility time of mice within 4 min after analysis.

Experimental results: The effect of spermidine on the immobility time in the tail suspension test of mice is shown in Table 3, and the experimental data are represented by Mean±SD. It can be seen from Table 3 that, consistent with the results of the forced swimming test in mice, the middle dose group (0.05g/kg) of the active part of spermidine and the low dose group (0.025g/kg) of the effective part of spermidine can reduce the tail suspension test of mice. Compared with the blank control group, there was a significant difference ( P<0.05 or P<0.01 ) in the immobility time in the control group, and it showed a certain dose dependence. The results of the forced swimming test in mice and the tail suspension test in mice showed that the active part of spermidine has obvious antidepressant effect, and its efficacy is equivalent to that of the classic antidepressant chemical drug venlafaxine.

Table 3 Effect of spermidine on immobility time in mouse tail suspension test (n=12)

Compared with the blank control group: * P<0.05 has a significant difference, ** P<0.01 has a very significant difference

In summary, the results of this experiment show that the effective part of spermidine can significantly reduce the immobility time in the animal model mice of behavioral hopelessness and depression. Therefore, it can be determined that the effective part of spermidine has significant antidepressant effect, and can be used to prepare medicine or health food for preventing and/or treating depression.

Example 8: Chronic Unpredictable Stress Model (CUMS)

Experimental instruments and reagents: animal behavior video analysis system (Chengdu Taimeng Technology Co., Ltd.), opening test box (self-made), thermometer, timer, animal gavage needle, 1000 ml measuring cylinder, stopwatch, clips, medical tape. Effective part of spermidine, sucrose (Tianjin Guangfu Technology Development Co., Ltd.), sodium carboxymethylcellulose (CMC-Na, Tianjin Kaitong Chemical Reagent Co., Ltd.), venlafaxine hydrochloride (Chengdu Kanghong Pharmaceutical Group Co., Ltd. Ltd).

Experimental animals and drug administration: male SD rats (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2016-0006), 180-220g, after 1 week of adaptive feeding, were randomly divided into 6 Group, 10 in each group. The dose of venlafaxine was 0.05g/kg, the high, medium and low doses of total spermidine effective fraction extracts were 34.6mg/kg, 17.3mg/kg, 8.65mg/kg, and the blank control group and model control group were given equal volume of distilled water. Animals in each group were given intragastric administration once a day, 10ml/200g each time, for 28 consecutive days according to body weight.

Establishment of CUMS depression model: stimulating factors include: fasting, water deprivation, restraint, tail pinching, ultrasonic stimulation, 4°C ice water bath, plantar electric shock, thermal stimulation, and circadian disturbance. One stimulus was randomly given every day, and each stimulus was no more than 4 times. Except for the blank control group, the rest of the groups were reared in single cages and implemented the modeling procedure, and the modeling time was 28 days. After the start of modeling, drugs were given by intragastric administration at the same time. The drug group was given corresponding concentrations of drugs, and the model group and blank control group were given equal volumes of distilled water.

Detection Indicator:

Body weight: the rats were weighed on the 0th, 7th, 14th, 21st and 28th day of the experiment.

Sugar water preference rate: measure the 1% sugar water consumption of rats at the beginning and end of modeling, and calculate the sugar water preference rate.

Open field test: Rats were placed in the open field box for 5 minutes, and the number of crossing grids and upright times of the rats within 4 minutes after recording.

Experimental results: the effect of the effective part of spermidine on the body weight, sugar water preference rate, open field test and other indicators of CUMS rats. It can be seen from Table 4, 5, and 6 that the effective part of spermidine can improve the stress caused by CUMS modeling. The slow weight growth of rats, decreased activity in the open field test, and decreased preference for sucrose sweetness indicate to a certain extent that the effective fraction of spermidine has a good antidepressant effect, and its pharmacological activity reaches or exceeds that of the antidepressant Western drug Venlafa pungent.

Table 4 Effects of modeling and administration on body weight changes in rats (x ± s, n=10, unit: g)

Compared with the model control group: ** p<0.01 , * p<0.05 Compared with the blank control group: ^## p<0.01 , ^# p<0.05

Table 5 Effects of modeling and drug administration on sugar water preference rate of CUMS rats (x ± s, n=10)

Compared with the model control group: ** p<0.01 , * p<0.05 Compared with the blank control group: ^## p<0.01 , ^# p<0.05

Table 6 Effects of the effective part of spermidine on the open field test of CUMS rats

Compared with the model control group: ** p<0.01 , * p<0.05 Compared with the blank control group: ^## p<0.01 , ^# p<0.05

In summary, the results of this experiment show that the effective part of spermidine can significantly reduce the immobility time in the animal model mice of behavioral hopelessness and depression, and improve the slow weight gain of rats caused by the stress of CUMS modeling, and the improvement in open field experiments. The decrease in activity and the preference for sucrose sweetness indicate that it has a good antidepressant effect to a certain extent, and its pharmacological activity reaches or exceeds that of the antidepressant western drug venlafaxine. Therefore, it can be determined that the effective part of spermidine has significant antidepressant effect, and can be used to prepare medicine or health food for preventing and/or treating depression.

Claims (5)

1. a kind of extracting method of antidepressant spermidine active component, it is characterised in that: specific step is as follows: taking dry safflower Or the safflower dregs of a decoction, 60-90% alcohol reflux extract 1-2 times, are concentrated under reduced pressure, concentrate is suppressed pressure in standby column progress in C18 quick Purifying, W1/W2 1-10, the W1 are sample quality to be separated in terms of mg, and W2 is the quality of C18 post separation material in terms of g； 10-100% ethyl alcohol or water elution are concentrated and dried up to spermidine effective part extract.

2. a kind of extracting method of antidepressant spermidine active component according to claim 1, it is characterised in that: obtained The spermidine substance that the three cumarin acyl groups that the spermidine effective part extract obtained contains 4 configurations replace, total content are 50% or more.

3. the spermidine active component that the extracting method of antidepressant spermidine active component according to claim 1 obtains The application of extract, it is characterised in that: the spermidine effective part extract is preparing antidepressant drug or health food In application.

4. the application of spermidine effective part extract according to claim 3, it is characterised in that: the antidepressant medicine Object is that pharmaceutically acceptable regular dosage form is made in the pharmaceutically acceptable auxiliary material of spermidine effective part extract addition.

5. the application of spermidine effective part extract according to claim 4, it is characterised in that: the dosage form is capsule Agent, tablet, powder, granule, gelling agent, sustained release agent, oral solution or pill.