A benzofuran compound in Eupatorium chinense and its use in treating depression

Abstract

The present invention discloses a benzofuran compound in Eupatorium chinense and its use in treating depression. The benzofuran compound is isolated from the Chinese medicinal material Eupatorium chinense and named: 10 R -2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran. The preparation method of the benzofuran compound uses the root of the Chinese medicine Eupatorium chinense as the raw material, and is obtained through steps of solvent extraction, activated carbon decolorization, macroporous adsorption column chromatography, and crystallization separation. This separation method is simple, effective, and has a high purity, and is applicable to industrial production. Through animal experiments on the benzofuran compound, behavioral data analysis such as the sucrose preference degree, tail suspension test, elevated plus maze test, forced swimming test, and open field test of mice is carried out. The benzofuran compound has a significant effect in treating depression, has a good efficacy in treating depression, and is expected to be developed into a new natural medicine for treating depression.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a preparation method of a traditional Chinese medicine extract 10R-2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran and its use in the treatment of depression. Background Art

[0002] The lifetime prevalence of adult depressive disorders in China is 6.8%, of which depression is 3.4%. Currently, the number of people suffering from depression in China is 95 million.

[0003] Currently, the treatment of depression mainly relies on Western medicine. Since the invention of the first-generation antidepressant drug, imipramine, in 1959, it has developed to the third-generation drug, vilazodone. However, analyzing from their mechanisms of action, these drugs have not stepped out of the framework of the classical "monoamine hypothesis", and only work on about 50% of patients. There are deficiencies such as low clinical cure rate, prominent residual symptoms, high recurrence rate, and easy to cause organ function disorders. In recent years, newly launched novel antidepressant drugs acting on various receptors (such as agomelatine and esketamine) have obvious characteristics in the treatment of depression. However, these novel drugs still face side effects brought about by the wide distribution of target receptors in the body, and there are also relatively serious adverse reactions. Therefore, it is still necessary to explore and develop novel antidepressant drugs from new sources. In recent years, domestic and foreign scholars have shifted their attention to extracts of Chinese herbal medicines, and many natural drugs with antidepressant effects have been newly discovered. Natural extracts such as morindabasyniosides and Hypericum perforatum extracts have been developed into clinical antidepressant drugs. Therefore, there is broad prospect in searching for natural antidepressant drugs from Chinese herbal medicines.

[0004] Eupatorium chinense is a plant of the genus Eupatorium in the Compositae family, also known as Du Xu Gong, Tu Niu Xi, Liu Yue Xue, etc. It is mainly distributed in Hubei, Hunan, Guangdong and other places. It is a commonly used herb of the Tujia ethnic group in western Hubei and western Hunan, and has the effects of clearing heat and detoxifying, soothing the liver and promoting blood circulation. The research group has long been committed to the study of the chemical constituents and pharmacological activities of Eupatorium chinense. It was previously found that its extract has good antidepressant activity (patent number: CN202010387432.8). A main component was obtained through separation and purification, which is compound 1 (10R-2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran) described in the present invention. After consulting the literature, it was found that there is no literature report on its therapeutic effect on depression. The present invention uses a classical antidepressant model method, namely the chronic unpredictable mild stress (CUMS) model, to establish a depression model in mice. This method is similar to the pathogenesis of human depression and is widely used in drug screening and drug mechanism research. Compound 1 described in the present invention was analyzed by behavioral data such as the sucrose preference degree, tail suspension test, elevated plus maze test, forced swimming test, open field test, etc. in mice. The antidepressant effect of this compound is significant, and it has a good effect in treating depression and is expected to be developed into a new natural antidepressant drug. The present invention patent discloses the preparation method of this compound and its use in treating depression. Summary of the Invention

[0005] The first object of the present invention is to provide a preparation method of compound 1 (10R-2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran), and the second object is to provide its use in the preparation of drugs for treating depression.

[0006] The present invention provides a preparation method of this compound, including the following steps:

[0007] Step A: Crush the roots of Eupatorium chinense, extract by heating under reflux with an aqueous alcohol solution, filter the extract, and concentrate under reduced pressure to obtain an extract.

[0008] Step B: Dissolve the extract obtained in Step A in an appropriate amount of water and load it onto an activated carbon chromatography column. First, wash it with water (the water selected from distilled water to avoid the entry of impurities) until it is colorless, and then elute it with ethanol. Collect the fractions and concentrate under reduced pressure to obtain an extract.

[0009] Step C: Dissolve the extract obtained in Step B in an appropriate amount of water and load it onto a macroporous adsorption resin chromatography column. Elute it with 90%-95% ethanol water (volume ratio), combine the eluates, and concentrate under reduced pressure to obtain an extract.

[0010] Step D: Completely dissolve the extract obtained in Step C in methanol, add pure water to make the volume fraction of the methanol solution 10-40, place it in a 4°C refrigerator overnight, centrifuge the crystal substance, and dry it to obtain the compound 1.

[0011] Furthermore, the alcohol aqueous solution in step A is 90-95% ethanol, preferably 95% ethanol.

[0012] Furthermore, the eluent in step B is 70%-90% ethanol.

[0013] Furthermore, the eluent in step C is 30%-40% ethanol.

[0014] The present invention also discloses the use of compound 1 in treating depression.

[0015] The advantages of the present invention are:

[0016] 1. The present invention discloses that compound 1 has a significant therapeutic effect on depression, provides a new medical use of compound 1, and opens up a new application field for it.

[0017] 2. Compound 1 has a significant effect in treating depression, which is mainly manifested by: increasing the activity distance and sugar water preference index of mice in the open field test, reducing their immobility time in the forced swim and tail suspension tests, and increasing their open arm exploration time in the elevated plus maze test. It greatly improves the depressive state of animals and has a significant anti-depressant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Compound 1 1 H-NMR spectrum.

[0019] Figure 2 Compound 1 13 C-NMR spectrum.

[0020] Figure 3 CD spectrum of compound 1.

[0021] Figure 4 Effects of compound 1 on behavioral activities of mice in the open field test. Note: *P<0.05, **P<0.01 compared with the normal control group; #P<0.05, ##P<0.01 compared with the model control group.

[0022] Figure 5 The exploration trajectories of mice in different groups in a confined space.

[0023] Figure 6 Effects of compound 1 on behavioral activities in the plus maze test of mice. Note: *P<0.05, **P<0.01 compared with the normal control group; #P<0.05, ##P<0.01 compared with the model control group.

[0024] Figure 7Effect of Compound 1 on the immobility time of mice in the forced swimming test. Note: Compared with the normal control group, *P<0.05, **P<0.01; compared with the model control group, #P<0.05, ##P<0.01.

[0025] Figure 8 Effect of Compound 1 on the immobility time of mice in the tail suspension test. Note: Compared with the normal control group, *P<0.05,

[0026] **P<0.01; compared with the model control group, #P<0.05, ##P<0.01.

[0027] Figure 9 Effect of Compound 1 on the sucrose consumption of mice. Note: Compared with the normal control group, *P<0.05, **P<0.01; compared with the model control group, #P<0.05, ##P<0.01. Detailed implementation manners

[0028] The following implementation cases are used to further explain and illustrate the present invention, but do not limit the protection scope of the present invention thereto.

[0029] Example 1: Preparation and structural analysis of Compound 1

[0030] The preparation method includes the following steps:

[0031] Step A: Grind the roots of Eupatorium chinense L. to 20-40 mesh, extract with 95% ethanol-water by heating under reflux 5 times, 2 hours each time, combine the extracts, filter, and concentrate under reduced pressure to obtain extract a;

[0032] Step B: Dissolve extract a in an appropriate amount of water and perform decolorization treatment using an activated carbon chromatography column. First, rinse with distilled water until colorless, and then elute successively with 70%, 75%, 80%, 85%, and 90% ethanol in a gradient manner. Elute 2 column volumes for each concentration, collect the eluates of each component and combine them, and concentrate under reduced pressure to obtain extract b;

[0033] Step C: Dissolve extract b in water, load it onto a macroporous adsorption resin chromatography column, elute with 40% ethanol-water (volume ratio), combine the organic phases, and concentrate under reduced pressure to obtain extract c;

[0034] Step D: Completely dissolve extract c in methanol, add 3 times the amount of pure water, place it in a refrigerator at 4°C overnight, centrifuge the crystal substances, and dry to obtain the said Compound 1.

[0035] Structural analysis of Compound 1:

[0036] Physical constants and spectral data of Compound 1: Pale yellow needle-shaped crystals, m.p. 185-187°C; [α] 25 D+10.1(c0.12,CH3OH); UV(CH3OH)λ max (logε)nm:250(3.06),350(1.85); CD(c 0.02mM,CH3OH)λ max (Δε):238(+0.91),381(-0.35);EI-MS m / z:250.1[M] + , C 13 H 14 O5. 1 H-NMR (400 MHz, DMSO-d6) δ H :12.35(1H,s,6-OH),8.18(1H,s,H-4),7.06(1H,s,H-7),6.71(1H,s,H-3),3.63(1H,dd,J=10. 7,6.0Hz,H-11a),3.54(1H,dd,J=10.7,6.0Hz,H-11b),2.69(3H,s,H-14),1.47(3H,s,H-12), see Figure 1 ; 13 C-NMR (100 MHz, DMSO-d6) δ c :205.2(C-13),163.8(C-8),160.0(C-6),159.1(C-2),124.9(C-4),121.6(C-9),117.2(C-5),102.8(C-3),99.2(C-7),71.5(C-10),68.5(C-11),27.8(C-14),24.0(C-12), see Figure 2 The present invention identifies the stereo configuration of C-10. The present invention tests the CD spectrum of the compound and finds a positive cotton effect at 238nm and a negative cotton effect at 381nm. Figure 3 , and the configuration of C-10 was determined to be R. Therefore, the compound was identified as 10R-2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran.

[0037] Example 2: Antidepressant Animal Experiment of Compound 1

[0038] 1. Experimental Materials

[0039] 1.1 Experimental Animals: 48 male C57BL / 6J mice (provided by the Animal Experiment Center of China Three Gorges University, license number SCXK(鄂)2022-0061), weighing 18–20 g.

[0040] 1.2 Experimental instruments: RS-12A ultrasonic cleaner (Rongshun Technology Instruments Factory, Ningbo, China), chemiluminescence imager (Tianneng Co., Ltd., Shanghai), FC5515R high-speed refrigerated centrifuge (Eppendorf Co., Ltd., Germany).

[0041] 1.3 Experimental drugs: Compound 1, sodium carboxymethylcellulose (Maclin USP grade), fluoxetine hydrochloride (Shanxi Qianyuan Pharmaceutical Group Co., Ltd., batch number: 202104, specification 20 mg)

[0042] 2. Experimental methods

[0043] 2.1 Animal grouping: After 3 days of adaptive feeding of mice, the mice were randomly divided into a normal control group, a model group, a positive control group (fluoxetine 10 mg / kg), a low-dose group of Compound 1 (4 mg / kg), a medium-dose group (8 mg / kg), and a high-dose group (16 mg / kg) according to body weight, with 8 mice in each group. Except for the normal control group, other groups were raised in isolation.

[0044] 2.2 Establishment of the depression model and drug administration: Except for the normal control group which was not treated otherwise and maintained normal drinking and eating, the mice in the model group and the drug administration groups were randomly selected to be exposed to any 1-2 of the following stimuli every day: the cage tilted at 45° for 24 h, wet bedding for 24 h, bedding deprivation for 24 h, food and / or water deprivation for 24 h, ice water swimming for 5 min, overnight strong light illumination, shaking the cage for 20 min, contact with foreign objects (rags, plastic bottles, plastic spoons, etc.) for 24 h, co-housing for 24 h, etc. The stimulation was continuous for 28 days, and the same kind of stimulation was not applied within 2 days after the stimulation was applied. At the same time, the mice in the model group and the drug administration groups were injected intraperitoneally with LPS every 3-4 days, with an injection dose of 0.1 mL / 10 g, and the mice in the normal group were injected with the same volume of normal saline. On the 8th day after the start of modeling, the mice in each group were given the corresponding drugs by gavage every day. The mice in the normal control group and the model group were given normal saline, the positive control group was given fluoxetine solution, and each dose group of Compound 1 was given the corresponding drug dose, and the drug administration was continuous for 28 days.

[0045] 2.3 Open field test: The mice were slowly placed in the center of a 40×40 cm open field. After the mice adapted to the field for 1 min, the activity status of the mice in the next 5 min was recorded, and the movement distance and movement situation of the mice were analyzed.

[0046] 2.4 Elevated plus maze test: The mice were gently placed in the center of the elevated plus maze device. After the mice adapted for 1 min, the activity and time of the mice in the open and closed arm areas in the next 5 min were recorded. The cumulative entry times into the open arms, the cumulative residence time in the open arms, the cumulative entry times into the closed arms, and the cumulative residence time in the closed arms were used as observation indicators. The proportion of the residence time in the open and closed arms was calculated.

[0047] 2.5 Forced swimming test: Add water to a transparent cylindrical water tank up to the scale line, ensuring the water temperature is around 25 °C. Place the mouse face-down into the water tank for the forced swimming test. Each mouse swims for 6 minutes, and recording starts after 1 minute of adaptation. Using the criteria that the mouse floats and its limbs do not move, record the total immobile time of the mouse in the subsequent 5 minutes starting from the 2nd minute.

[0048] 2.6 Tail suspension test: Fix the mouse at a position 2 cm from the tip of its tail to a special mouse tail suspension box with medical tape, making its head 15 cm from the ground. Each mouse is suspended for 6 minutes, and recording starts after 1 minute of adaptation. Using the criteria that the mouse gives up struggling and its limbs hang in the air without moving, record the total immobile time of the mouse in the subsequent 5 minutes starting from the 2nd minute.

[0049] 2.7 Sucrose consumption test: 48 hours before the test, place water bottles containing normal drinking water and 1% sucrose solution on the mouse cage. After 24 hours, swap the positions of the water bottles to let the mice get used to the sucrose solution for 24 hours. Before the test, fast and withhold water from the mice for 12 hours. Then prepare one bottle of normal drinking water and one bottle of 1% sucrose water, weigh them, and place them on the mouse cage. After 12 hours, gently remove the water bottles, weigh them, calculate and statistically analyze the consumption of normal drinking water and 1% sucrose water, and calculate the sucrose preference: sucrose consumption / (sucrose consumption + pure water consumption) × 100%, and the sucrose preference value can be obtained by calculation.

[0050] 2.8 Statistical analysis: The data is statistically analyzed and a between-group difference test is performed. The data is expressed as X±S. One-way analysis of variance (ANOVA) using SPSS statistical software is used for between-group differences, with P<0.05 indicating significant differences.

[0051] 3. Experimental results

[0052] 3.1 Effect of Compound 1 on the total movement distance of mice in the open field test

[0053] In the open field test, the total movement distance of the mice in the model group was lower than that in the normal control group, with significant differences (P<0.01), successfully establishing a mouse model of depression. The total movement distances of the mice in the positive control group and the low, medium, and high-dose groups of the administration group were all higher than that in the model group, with significant differences (P<0.05), all increasing the exploration time of the mice in the open field, and the effects of the low, medium, and high-dose groups were all better than those of the positive drug fluoxetine group. Analyzing from the data, the low-dose group had the best effect, as shown in Figure 4 . The mouse movement trajectory map ( Figure 5 ) shows that the mice in the model group preferred to move in the edge area of the test box, and the number of stops in the middle was significantly reduced compared with the control group mice.

[0054] 3.2 Effect of Compound 1 on the behavioral activities of mice in the elevated plus maze test

[0055] In the elevated plus-maze test, compared with the normal control group, the proportion of the number of times the mice in the model group entered the open arms decreased, and the difference was significant (P < 0.01), successfully establishing a mouse model of depression. The proportion of the number of times the mice in the positive control group and the low, medium, and high dose groups of the administration group entered the open arms was significantly higher than that in the model group, and the difference was significant (P < 0.05). Among them, the effects of the low dose group and the high dose group were comparable to those of the positive drug fluoxetine group. Analyzed from the data, the high dose group had the best effect, as shown in Figure 6 .

[0056] 3.3 Effects of Compound 1 on the immobility time of mice in the forced swimming test

[0057] In the forced swimming test, compared with the normal group, the immobility time of the mice in the model group in water increased significantly, and the difference was significant (P < 0.01), successfully establishing a mouse model of depression. The immobility time of the mice in the positive control group and the low, medium, and high dose groups of the administration group in water was lower than that in the model group, and the difference was significant (P < 0.05 or P < 0.01). Analyzed from the data, the effects of the high dose group and the low dose group were better than those of the positive drug group. Among them, the low dose group had the best effect, as shown in Figure 7 .

[0058] 3.4 Effects of Compound 1 on the immobility time of mice in the tail suspension test

[0059] In the tail suspension test, compared with the normal group, the immobility time of the mice in the model group increased significantly, and the difference was significant (P < 0.01), successfully establishing a mouse model of depression. The immobility time of the mice in the positive control group and the low, medium, and high dose groups of the administration group in water was lower than that in the model group, and the difference was significant (P < 0.01). The effects of the high dose group and the low dose group were better than those of the positive drug group. Among them, the low dose group had the best effect, as shown in Figure 8 .

[0060] 3.5 Effects of Compound 1 on the sucrose consumption of mice

[0061] In the sucrose preference test, the sucrose preference value of the mice in the model group was significantly lower than that of the blank control group, and the difference was significant (P < 0.01), successfully establishing a mouse model of depression. The sucrose preference values of the mice in the positive control group and the low, medium, and high dose groups of the administration group were higher than that in the model group, and the difference was significant (P < 0.05). The effects of the low and medium doses were better than those of the positive drug fluoxetine group. Among them, the low dose group had the best effect, as shown in Figure 9 .

[0062] The sucrose preference rate of the depression model mice decreased significantly. The immobility time in the tail suspension test and forced swimming test increased significantly. The exploration times in the open field test and elevated plus maze decreased significantly, indicating that the depression model mice had anhedonia, a significantly decreased hedonic behavior index, increased despair behavior, decreased spontaneous activity, and decreased curiosity and exploration of the new environment. Thus, a depression model mouse was successfully constructed.

[0063] The depression model mice were intervened and treated by intragastric administration of Compound 1. The behavioral data of the mice were collected and analyzed. Compared with the model control group, both Compound 1 and fluoxetine had certain antidepressant effects on the depressed mice. In the open field test, the low, medium, and high dose groups of Compound 1 all increased the exploration time of the mice in the open field, and the effects were better than those of the positive drug fluoxetine group. Analyzed from the data, the low dose had the best effect. In the elevated plus maze test, the low, medium, and high dose groups of Compound 1 all increased the exploration times of the mice in the open arms, which were comparable to those of the positive drug fluoxetine group. Analyzed from the data, the high dose group had the best effect. In the forced swimming test, the immobility time of the mice in the low, medium, and high dose groups of Compound 1 decreased significantly, which was comparable to that of the positive drug fluoxetine group. Analyzed from the data, the high dose and low dose groups had better effects than the positive drug group, and the low dose group had the best effect. In the tail suspension test, the immobility time of the mice in the low, medium, and high dose groups of Compound 1 decreased significantly. Analyzed from the data, the high dose and low dose groups had better effects than the positive drug group, and the low dose group had the best effect. In the sucrose preference test, the low, medium, and high dose groups of Compound 1 all increased the sucrose preference rate of the mice, and the effects of the low and medium doses were better than those of the positive drug fluoxetine group, and the low dose group had the best effect. Comprehensive analysis of various behavioral data showed that the low dose group of Compound 1 had the best effect on improving the depressive behavior of the depression model mice induced by LPS combined with CUMS. The data were comparable to those of the positive drug, and some data were even better than those of the positive drug fluoxetine.

[0064] In summary, the present invention provides the use of Compound 1 in the preparation of drugs for treating depression. In the pharmacological experiments of the present invention, it was found that in the behavioral experiments of depression model mice, the depressive symptoms of the mice were significantly improved, mainly manifested as an increase in the activity distance and sucrose preference index of the mice in the open field test, a decrease in the immobility time of the mice in the forced swimming and tail suspension tests, and an increase in the exploration time of the mice in the open arm area of the elevated plus maze test. The depressive behavior was improved in various behavioral level tests, indicating that it had an obvious therapeutic effect on the depression model mice and was expected to be developed into a new natural antidepressant drug.

[0065] Although the present invention has been described in detail above through general descriptions, specific implementation examples, and activity experiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Use of a benzofuran compound in the preparation of a drug for treating depression, the benzofuran compound having a molecular formula of C 13 H 14 O5 and having the following structure: , named: 10 R -2-(10-methyl-10,11-dihydroxy)-5-acetyl-6-hydroxybenzofuran.

2. The application according to claim 1, wherein The preparation steps are as follows: A. Solvent extraction: The roots of *Eupatorium chinense* are crushed and then extracted by heating under reflux with an organic solvent, followed by filtration and concentration under reduced pressure to obtain extract a; B. Activated carbon decolorization: Extract a is dissolved in water and then decolorized using an activated carbon chromatography column, washed with water and then with alcohol, the fractions are collected, and concentrated under reduced pressure to obtain extract b; C. Macroporous adsorption column chromatography separation: Extract b is dissolved in water, loaded onto a macroporous adsorption resin chromatography column, washed with alcohol, the organic phases are combined, and concentrated under reduced pressure to obtain extract c; D. Separation by crystallization method: It is completely dissolved in a methanol solution and then cooled for crystallization to obtain Compound 1.

3. The application according to claim 2, characterized in that, The organic solvent in Step A is ethanol with a concentration of 90 - 95%.

4. The application according to claim 2, wherein The alcohol used in the alcohol washing in Step B is ethanol with a concentration of 70% - 90%.

5. The application according to claim 2, wherein The alcohol used in the alcohol washing in Step C is ethanol with a concentration of 30% - 40%.

6. The application according to claim 2, characterized in that, The concentration of the methanol solution in Step D is 10 - 40%.

7. According to the application described in Claim 1, the dosage form of the drug for treating depression is selected from any one of liquid preparations, solid preparations, sprays or aerosols.

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