WO2006000158A1

WO2006000158A1 - The use of laetispicine for manufacture of pharmaceutical composition

Info

Publication number: WO2006000158A1
Application number: PCT/CN2005/000912
Authority: WO
Inventors: Sheng-Li Pan; Fu-Gang Qian; Jun Wang; Feng-Yan Sun; Jing Xie; Yi-Ci Shao
Original assignee: Fudan University
Priority date: 2004-06-25
Filing date: 2005-06-24
Publication date: 2006-01-05
Also published as: CN1332656C; CN1593399A; WO2006000158A8

Abstract

The present invention discloses the use of laetispicine for manufacture of pharmaceutical composition for certain diseases caused by central nerves system monoamine neurotransmitter reuptake disorder. The isotope labeling central nerves system monoamine neurotransmitter reuptake test certifies the inhibiting activity of laetispicine to the reuptake of 5-HT, NE AND DA by rats’ synaptosomes. The present laetispicine can be used as raw material to prepare pharmaceutical composition which can inhibit the reuptake of central nerves system monoamine neurotransmitter, in order to treat the related diseases caused by reuptake of central nervessystem monoamine neurotransmitter.

Description

Application of compound macrophyllin in preparing pharmaceutical composition

The invention belongs to the field of traditional Chinese medicine and relates to an application of an amide compound daidzein isolated from the plant Eucalyptus urophylla in the preparation of a pharmaceutical composition. Specifically, it relates to the use of macrophyllin for the preparation of a pharmaceutical composition for inhibiting a central nervous system monoamine-based re-uptake-related disease. Background technique

With the continuous improvement of people's quality of life and health, neurological, mental, cardiovascular and endocrine systemic diseases have become the most common diseases in the world that endanger human health, and have already brought a heavy burden to human society. Serotonin (5-HT), dopamine (DA) and norepinephrine (NE) are the most important monoamine neurotransmitters in the central nervous system. Their levels in the brain control human emotions, emotions, regulate cardiovascular and endocrine functions, and are related to human body temperature, sleep, food intake, and motor systems. The main metabolic pathway after the release of monoamine neurotransmitters is reuptake, thereby controlling the normal content of synaptic gaps in this brain. Abnormalities in reuptake of monoamine transmitters result in a decrease in 5-HT, NE, and DA levels in the central nervous system, leading to disorders in the nervous system, cardiovascular system, learning and memory, endocrine, and motor systems. These include: manic or depressive disorders, sleep disorders, pain, dyskinesia, and sexual dysfunction. At present, various types of related chemical drugs still cannot meet the needs of increasing clinical treatment.

According to reports, the discovery of antidepressants and drugs for the treatment of mental disorders in natural medicines in China has formed a research hotspot. Studies have found that extracts extracted from ethanol extracted from alcoholic Chinese medicine large leaves have analgesic, sedative and antidepressant effects. Summary of the invention

It is an object of the present invention to provide a use of an amide compound, Laetispicine, isolated from a plant leaf lobe, for the preparation of a pharmaceutical composition. Specifically, it relates to a novel use of the compound daunsin in the preparation of a pharmaceutical composition for reuptake a disease in which the monoamine neurotransmitters 5-HT, NE and DA of the central nervous system are inhibited.

The present invention extracts and isolates Laetispicine from Piper laetispicum C. DC., a pepper plant, by using an isotope-labeled central nervous system monoamine transmitter reuptake test. It was tested to confirm that it inhibits the reuptake of serotonin, dopamine and norepinephrine by synaptosomes in the rat brain. The compound macrophyllin of the present invention can be used as a raw material to prepare a pharmaceutical composition for inhibiting a disease related to reuptake of a monoamine neurotransmitter in the central nervous system, or to inhibit a central nervous system monoamine containing at least one excipient. A pharmaceutical composition for re-intake-related diseases, including capsules, tablets, injections, oral solutions, inhalants, sustained release agents and the like. It is used to treat related diseases caused by re-uptake of monoamine transmitters in the central nervous system.

The raw medicinal material used is the pepper family

C. DC. Roots, rhizomes, stems, leaves, flowers or fruits.

The related diseases include depression caused by serotonin reuptake or decreased synaptic 5-HT content, obsessive-compulsive disorder, phobia, anxiety and hypertension, insomnia or pain; dopamine Depression, anxiety, obsessive-compulsive disorder, motor dysfunction, neurodegenerative diseases (Parkinson's disease, tremor palsy), and sexual dysfunction caused by decreased re-intake or decreased synaptic DA content; Cardiovascular depression caused by adrenaline reuptake or decreased synaptic NE content caused by manic depressive disorder, anxiety, narcolepsy, learning and memory dysfunction, Alzheimer's disease, eating abnormalities, obesity and autonomic dysfunction Systemic diseases (such as: bradycardia caused by low sympathetic nerve hypotension, hypotension) and sensory dysfunction caused by decreased sympathetic function (such as painlessness), abnormal body temperature regulation.

The present invention relates to Laetispicine chemical name: N-isobutyl-l l-(3,4-methy lend-ioxyphenyl)-2E, 4E, 9E-undecatrienamide, from the pepper-grown plant Big-mouth ten Piper laetispicum C The separation in DC is obtained. The separation is extracted by the following method:

After the raw medicinal material was pulverized, it was heated to reflux with 95% industrial alcohol, filtered, cooled, and the residue was subjected to secondary reflux with 95% industrial alcohol, filtered under cooling, combined with a filtrate, and concentrated to dryness under reduced pressure to give an ethanol extract. Add appropriate amount of water to the ethanol extract, extract 1:1 with petroleum ether, benzene, chloroform and ethyl acetate, heat to reflux, cool the layers, combine the organic phases, and concentrate under reduced pressure to obtain petroleum ether extract and benzene dip. Cream, chloroform extract, ethyl acetate extract. The ethyl acetate extract was subjected to silica gel column chromatography, and eluted with a gradient of ethyl hexanoacetate to give six fractions: Fr. I Fr. II, Fr. IIL Fr. IV, Fr. V, Fr. In VL, Fr. IV was further subjected to column chromatography, and gradient elution with petroleum ether-acetone was carried out to collect four fractions: Fr.IV_l, Fr.IV-2, Fr.IV_3, Fr.IV-4, Among them, Fr. IV-3 precipitated colorless needle crystals, and crystals were obtained by filtration. The series of spectra of HRMS, EIMS, ' ³ CR, DEPT, 'HNMR, HMQC, HMBC and COSY proved to be N-isobutyl- 11 -(3,4 -methylendioxyphenyl)-2E, 4E, 9E-undecatrienamide, ie large. Peony (laetispicine). The structure is as follows:

The compound of the present invention, lutein, is tested by isotopically labeled central nervous system monoamine neurotransmitter reuptake assay, see drawing: Laetispicine antidepressant mechanism study confirmed that it can inhibit synaptosome pairs in rat brain 5 Re-uptake of serotonin, dopamine and norepinephrine. DRAWINGS

FIG 1 is a large leaf Jun inhibitory effects of compounds in rats by the central nervous system labeled H 5- HT ³ reuptake. FIG 2 is a large leaf Jun inhibitory effects of compounds in rats by the central nervous system H ³ labeled norepinephrine reuptake. '

3 is a large leaf inhibitory effects of compounds in rats by Jun H ³ labeled CNS dopamine reuptake. detailed description

Example 1 Extraction and separation of large leaf flavonoids

After pulverizing 1000 g of raw medicinal material, adding 6 times of 95% industrial alcohol and heating and refluxing for 2 hours, cooling and filtering, the residue is added with 4 times of 95% industrial alcohol for secondary reflux, cooling and filtering, and combining the two filtrates. The organic layer was concentrated to dryness under reduced pressure to give 50 g of ethanol extract. Add an appropriate amount of water to the ethanol extract, and extract 1:1 with five times of petroleum ether, benzene, chloroform and ethyl acetate. Heat the reflux several times, cool the layers, combine the organic phases, and concentrate under reduced pressure. 0.5 g of petroleum ether extract, 8 g of benzene extract, 6 g of chloroform extract, and 10 g of ethyl acetate extract were obtained.

10 g of ethyl acetate extract was subjected to silica gel column chromatography eluting with a gradient of ethyl acetate (5: 1-3.5: 1.5) to give six fractions: Fr. I, Fr. II, Fr .m, Fr.IV, Fr.V, Fr.VL Further Fr. IV column chromatography, gradient elution with petroleum ether-acetone (6: 1~3: 1), four fractions were collected: Fr.IV-1, Fr.IV-2, Fr.IV-3, Fr.IV-4' wherein Fr. IV-3 precipitates colorless needle crystals, which are filtered to obtain crystals 300mg, by HRMS, EIMS, ¹³ CNMR, DEPT , NMR, HMQC, HMBC, COSY A series of spectra proved to be N-isobutyl-ll-(3,4-methylendioxyphenyl)-2E, 4E, 9E-undecatrienamide, 艮卩大口十素素 (laetispicine).

Example 2 Isotope-labeled central nervous system monoamine transmitter reuptake inhibition test

150-250 g Sprague-Dawley male rats were sacrificed, the neck was removed, the brain was removed, and the brain area was removed. The hypothalamus was used for 5-HT and NE reuptake inhibition experiments. The striatum was used for DA reuptake inhibition experiments, weighing, 25 mL/g ice-cold 0.32 M sucrose solution was added and the paddle was homogenized in an ice bath. The homogenate was centrifuged at 1000 rpm for 10 min, and the precipitate was discarded. The supernatant was taken and centrifuged at 10,000-17,000 rpm for 20 min. The precipitate is the synapse. The above 0.32 M sucrose solution was suspended at a ratio of lOm! Jg (based on the wet weight of the brain tissue). The protein content was determined. spare.

H ³ labeled monoamine neurotransmitters, diluted 200-fold in Krebs solution freshly prepared. Take 800 μL + 20 μL of drug (or solvent) and mix well. Add 25 μL of synaptosome suspension and start timing. After 5 min, it was filtered on Whatman GFB filter paper and washed with ice-cold Krebs solution. After the filter paper is dried, pour in the scintillation fluid and measure the number of flashes per minute (CPM). Under the same conditions, the CPM value of 37 degrees Celsius minus the CPM value of 0-4 degrees Celsius is the re-uptake value of the monoamine transmitter in the central nervous system. The inhibition of the reuptake of monoamine transmitters by the drug was examined.

The above sucrose solution contains sucrose 0.32 M, EDTA 0. 0001 M, glucose lmg/mL, tris adjusted pH to 7.4.

The bicarbonate Krebs reaction solution is composed of

Composition final concentration m mol/L)

NaCl 130

KC1 2.74

NaH ₂ P0 ₄ 0.70

MgCl ₂ 0.50

NaHC0 ₃ 30

Glucose 10

EDTA 0.171

Iproniazid 0.10

pH 7.3

Among them: (1) . iproniazid is a monoamine oxidase inhibitor (MAOI), which can be replaced by others, such as pargylkie ₀

(2) . The pH is usually adjusted with tris.

4.95% 0 ₂ -5% C0 _{2 is} saturated.

The scintillation liquid has a composition of

PPO 5g,

POPOP 0.2g

Add xylene to 1000ml. Table 1 is a large leaf Yun factor on central nervous system in rats ^{H: i} labeled 5- HT reuptake inhibition results. The membrane CPM value is the average of the three scintillation counts after subtracting the background.

Table 2 is a large leaf central nervous system drugs in rats by H ³ labeled NE reuptake inhibition results. The membrane CPM value is the average of the three scintillation counts after subtracting the background.

Table 3 shows the results of inhibition of H ³ labeled DA reuptake by the central nervous system in rats. The membrane CPM value is the average of the three scintillation counts after subtracting the background.

ο

Table 1

Table 2.

table 3.

Synaptosome site drug concentration membrane CPM (0 °C) membrane CPM (37Ό) inhibition rate (%) striatum Control 19417 122065

Striatum ^{1 X 10 "8 mol / L} 19680 113552 8. 55 1544297721 22.76 striatum striatum ^{1 X 10- 6 mol / L 18145} 78852 40. 86 striatum 1 X 10" ⁵ mol / L 19440 73467 47. 37 striatum ^{1 X 10- 4 mol / L 20723} 68828 53. 14

Claims

Rights request

1. Use of a compound cerulein for the preparation of a pharmaceutical composition for inhibiting central nervous system monoamine neurotransmitter re-ingtake.

2. The use according to claim 1, wherein said compound daidzein is extracted and separated by the following method,

After pulverizing the raw medicinal material, adding 95% industrial alcohol and heating and refluxing for 2 hours, cooling and filtering, the residue was added with 95% industrial alcohol for secondary reflux, cooled and filtered, and the filtrate was combined twice, and concentrated to dryness under reduced pressure. Ethanol extract. Add equal amount of water to the ethanol extract, extract 1:1 with petroleum ether, benzene, chloroform and ethyl acetate, heat to reflux, cool the layers, combine the organic phases, and concentrate under reduced pressure to obtain petroleum ether extract and benzene. The extract, the chloroform extract, and the ethyl acetate extract, wherein the ethyl acetate extract was subjected to silica gel column chromatography, and eluted with a gradient of ethyl cyclohexane-acetate (5:1 to 3.5:1.5) to obtain a fraction: Fr. I, Fr. II, Fr.nL Fr. IV, Fr. V, Fr. VL wherein Fr. IV is further subjected to column chromatography, eluting with petroleum ether-acetone 6:1~3:1 gradient, collecting stream Points: Fr. IV-1, Fr.IV-2, Fr.IV-3, Fr.IV-4, wherein Fr. IV-3 precipitates colorless needle crystals, and the crystals are filtered, ie, macrophylto.

The use according to claim 1 or 2, characterized in that the compound daidzein is used as a raw material to prepare a capsule, a tablet, an injection, an oral solution, an inhalant or a sustained release agent.

4. The use according to claim 2, wherein the raw material is root, rhizome, stem, leaf, flower or fruit of Piper _ZseiJ ic C. DC.

5. The use of claim 1, wherein the central nervous system monoamine neurotransmitter is serotonin or dopamine or norepinephrine.

6. The use according to claim 1, wherein the central nervous system monoamine neurotransmitter re-uptake-related disease is depression caused by a decrease in serotonin reuptake or a decrease in 5-HT content in the synaptic cleft, obsessive-compulsive disorder, Phobia, anxiety or high blood pressure, insomnia and pain symptoms.

7. The use according to claim 1, wherein said central nervous system monoamine neurotransmitter reuptake-related disease is depression, anxiety disorder, obsessive-compulsive disorder, exercise caused by decreased dopamine reuptake or synaptic interstitial M content. Dysfunction, neurodegenerative diseases including Parkinson's disease, tremor hemp 痹, or sexual dysfunction.

The use according to claim 1, wherein said central nervous system monoamine neurotransmitter reuptake-related disease is manic depressive disorder, anxiety caused by norepinephrine reuptake or decreased synaptic NE content. Cardiovascular diseases caused by narcolepsy, learning and memory dysfunction, Alzheimer's disease, eating abnormalities, obesity or autonomic dysfunction, including bradycardia caused by sympathetic hypoactivity, hypotension, or decreased sympathetic function Symptoms of sensory function include painlessness or abnormal body temperature regulation.