CN109223816B

CN109223816B - Application of eleutheroside D in preparation of anti-depression drug

Info

Publication number: CN109223816B
Application number: CN201811383052.6A
Authority: CN
Inventors: 颜露; 韦敏; 宋萍萍; 钱怡云; 郑生智
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2018-11-20
Filing date: 2018-11-20
Publication date: 2021-10-26
Anticipated expiration: 2038-11-20
Also published as: CN109223816A

Abstract

The invention discloses an application of eleutheroside D in preparing an anti-depression drug. Eleutheroside D has remarkable effect in treating depression. The eleutheroside D is prepared into oral administration, transdermal administration or injection administration for treating depression.

Description

Application of eleutheroside D in preparation of anti-depression drug

Technical Field

The invention belongs to the technical field of medicines, relates to medical application of eleutheroside D, and particularly relates to application of eleutheroside D in a medicine for effectively treating depression.

Background

Depression is a common mood disorder, is characterized by significant and persistent mood depression as a major clinical feature, has globally increased prevalence rates, and is expected to be the second largest disease after coronary heart disease by 2020. The incidence of depression in China is about 4%, depression patients cannot adapt to the society, are slow in thinking, poor in memory and the like, are high-risk groups with suicide, self-disability and abuse, and form a great social health crisis. Moreover, clinical studies find that the depression coexists with various diseases such as Parkinson's disease, diabetes and the like, and serious neuropsychiatric burden is brought to patients. Therefore, depression has become a prominent social problem, and the research on depression has become a hot spot of concern at home and abroad.

At present, the research at home and abroad considers that the pathogenesis of depression is complex and is related to various factors such as heredity, environment, society and the like. The following hypotheses are mainly involved: 1 neurotransmitter hypothesis, central 5-hydroxytryptamine nervous system dysfunction, 5-hydroxytryptamine (Serotonin, 5-HT) in synaptic cleft, Norepinephrine (NE), and Dopamine (DA) levels are among the major pathogenesis of depression. 2, the neurotrophic factor hypothesis, Nerve Growth Factor (NGF), Brain-derived neurotrophic factor (BDNF), Glial cell line-derived neurotrophic factor (GDNF), etc. have important effects on the growth, differentiation and maintenance of neuron function. The reduction of expression level of hippocampal neurotrophic factor and its receptor can reduce neurogenesis and neuron survival, and cause depression. The neuroendocrine hypothesis, in which the hypothalamus-pituitary-adrenal (HPA) axis is hyperactive, and cortisol is secreted too much, causing damage to neurons in the hippocampus, which in turn causes depression.

Modern medicine is currently taking the elevation of synaptic cleft neurotransmitter levels as the main strategy for the treatment of depression. Typical anti-depression drugs are mainly: tricyclic antidepressants (TCAs), such as imipramine, and the like; selective Serotonin Reuptake Inhibitors (SSRIs), such as fluoxetine, and the like; monoamine oxidase inhibitors (MAOI), such as phenelzine, and the like. These drugs have various side effects and high tolerability, and there is a delay in treatment time, suggesting that it is difficult to achieve good effects for the prevention and treatment of depression only by regulating neurotransmitter levels. The neurotrophic factor hypothesis considers that the onset of depression is closely related to the damage of neurogenesis, and the expression of the neurotrophic factor and a receptor thereof is regulated up, so that the neurogenesis is promoted to effectively relieve the depression, which also helps explain that the antidepressant drug needs several weeks to recover the neurogenesis to exert the curative effect. Therefore, neurotransmitter and neurotrophic factors are used as action targets, and new anti-depression drugs are searched for to effectively prevent and treat depression.

Eleutheroside D (eleutheroside D) is a triterpene compound mainly distributed in plant of Araliaceae family. Eleutheroside D is white needle crystal, and has molecular formula: c₃₄H₄₆O₁₈Molecular weight: 742.72 has the following structure, and has effects of resisting platelet aggregation, relieving fatigue, and relieving inflammation. At present, the application of eleutheroside D in preparing antidepressant medicaments is not seen.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of eleutheroside D in preparing anti-depression drugs. The eleutheroside D has the following structure:

the invention relates to an application of eleutheroside D in resisting depression, which can remarkably regulate the level of neurotransmitter, promote expression of neurotrophic factors and receptors thereof, and has the characteristics of safe use, long-term use and the like when being used for the application. Therefore, the eleutheroside D can be applied to the preparation of anti-depression drugs, and the drugs have the application of treating depression.

Eleutheroside D can be obtained from commercial sources or prepared by the prior art.

The eleutheroside D applied by the invention can be prepared into a medicament with any pharmaceutically allowed auxiliary material or pharmaceutical excipient, and the preparation can be any pharmaceutically allowed dosage form, including but not limited to granules, tablets, granules, soft capsules, dripping pills, ointments or injections.

The administration form of the medicament provided by the invention mainly comprises oral administration, transdermal administration or injection administration.

The dose of eleutheroside D in the present invention varies depending on the condition, body weight, administration method, etc. of the patient. For example, the dosage is 0.1-300 mg/(kg.d) for non-oral administration, intravenous administration and intestinal administration, and 0.4-1200 mg/(kg.d) for oral administration.

The invention adopts the maximum tolerance method to carry out the gavage test on the mice, and the result shows that the mice are healthy and have no abnormal reaction after the action, the anatomy has no obvious pathological changes, the organ index and the serum index have no obvious difference from the normal group, and the eleutheroside D has no obvious difference on the oral LD of the mice₅₀>15 g/kg, which shows that the eleutheroside D has no toxic effect on mice and is safe.

Compared with the prior art, the invention has the beneficial effects that: eleutheroside D can significantly shorten forced swimming and tail suspension accumulation immobility time of normal mice, has significant antidepressant effect on chronic stress depressed rats, can significantly improve 5-hydroxytryptamine, norepinephrine, 5-oxindole acetic acid/5-hydroxytryptamine level and striatal dopamine level of depressed rats, and significantly up-regulates neurotrophic factors and receptor expression of depressed rats in hippocampal, and acute toxicity experiments show that eleutheroside D belongs to nontoxic grade. Therefore, the eleutheroside D can be applied to the preparation of anti-depression drugs. The invention provides a new clinical application of eleutheroside D and enlarges the application range of the eleutheroside D.

Drawings

FIG. 1 is a graph showing the results of a forced swimming test of a mouse.

FIG. 2 is a graph showing the results of the tail suspension test of mice.

FIG. 3 is a graph of the results of weight measurement of rats with chronic stress depression.

FIG. 4 is a graph showing the results of the carbohydrate preference test in rats with chronic stress depression.

FIG. 5 is a graph showing the results of a forced swimming test in rats with chronic stress depression.

FIG. 6 is a graph showing the results of neurotransmitter detection in chronically stressed depressed rats.

FIG. 7 is a graph showing the result of detecting neurotrophic factor in hippocampal region of rats with chronic stress depression.

FIG. 8 is a graph showing the results of detecting neurotrophic factor receptors in hippocampal regions of rats with chronic stress depression.

Detailed Description

The present invention will be further described with reference to specific examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental animals are purchased from Changzhou Kavens experimental animals Limited company (production license number: SCXK (Su) 2016-. The eleutheroside D reference substance is prepared by self, has purity of more than or equal to 98%, and is dissolved in water to prepare administration concentration (0.5, 1.5, 4.5 mg/mL or 3.33, 10, 30 mg/mL).

EXAMPLE one Effect of Eleutherobin D on cumulative immobility time in forced swimming of mice

ICR mice, male, were bred for 7 days to adapt to the new environment. Mice were divided into a blank control group, an eleutheroside D group [5, 15, 45 mg/(kg.d) ], a imipramine group [30 mg/(kg.d) ], a levodopa group [60 mg/(kg.d) ] and a galantamine group [1.5 mg/(kg.d) ], 12 mice each, administered by gavage at 10:00 am daily for 7 days. After 1 h of the last administration, a forced swimming test was performed. The test was performed in 2 days: the mice were allowed to swim in 22 + -2 deg.C deep water for 15 min on day 1, and after 24 h, forced swimming was performed for 6 min under the same conditions, and the cumulative immobile time for swimming within 4 min after counting (i.e., the mice stopped struggling or the mice were floating, and the limbs slightly moved to keep the head on the water surface) was shown in FIG. 1. The result shows that the accumulative immobility time of the forced swimming of the mice after the positive drug and the eleutheroside D [5, 15, 45 mg/(kg.d) ] are obviously different from the model group (p <0.05 or p < 0.01).

EXAMPLE two Effect of Eleutherobin D on cumulative immobility time of mouse Tail suspension

ICR mice, male, were bred for 7 days to adapt to the new environment. Mice were divided into a blank control group, an eleutheroside D group [5, 15, 45 mg/(kg.d) ], a imipramine group [30 mg/(kg.d) ], a levodopa group [60 mg/(kg.d) ] and a galantamine group [1.5 mg/(kg.d) ], 12 mice each, administered by gavage at 10:00 am daily for 7 days. The tail suspension test was performed 1 h after the last administration. The position about 1 cm away from the tail tip of the mouse is pasted on a bracket of a tail suspension box (25 multiplied by 30 cm) by using an adhesive tape to enable the mouse to be in an upside-down suspension state, the suspension time is 6 min, the accumulated dead time of tail suspension in the 6 min of the mouse is counted (namely the mouse stops struggling or has no movement), and the result is shown in figure 2. The result shows that the mouse tail suspension accumulation immobility time after the positive drug, the eleutheroside D [5, 15, 45 mg/(kg.d) ], the levodopa and the galantamine act is obviously different from the model group (p is less than 0.05 or p is less than 0.01).

EXAMPLE III Effect of Eleutherobin D on body weight in chronically stressed depressed rats

SD rats, male, were kept for 7 days to adapt to the new environment. Animals in each group, except the placebo group, were given a series of chronic stress stimuli of (1) food deprivation for 24 h; (2) water deprivation for 24 h; (3) exposure to an empty bottle for 1 h; (4) cage tilt (45 °) 7 h; (5) illuminating overnight; (6) a sewage cage (200 mL of water and 100 g of sawdust are added in the cage) for 24 hours; (7) forced swimming for 6 min at 8 deg.C; (8) physical limitation for 2 h; (9) exposed to a foreign object (e.g., a plastic sheet) 24 h. The above stress stimuli were randomly scheduled to be completed within 1 week and repeated for at least 4 weeks. And after the modeling is finished, evaluating the depression degree of the model through sugar water preference degree and weight tests. Rats were divided into a blank control group, an eleutheroside D group [3.33, 10, 30 mg/(kg.d) ], a imipramine group [20 mg/(kg.d) ], a levodopa group [40 mg/(kg.d) ], and a galantamine group [1 mg/(kg.d) ], each group of 12 animals administered by gavage at 10:00 am daily for 4 weeks. Body weight measurements were made for each group of rats after the modeling was completed and the dosing was completed, and the results are shown in fig. 3. The results show that the model group has significant difference (p < 0.01) with the normal control group, and the weight of the patient after 4 weeks of administration of the positive drug and eleutheroside D [3.33, 10, 30 mg/(kg.d) ] has significant difference (p < 0.05) with the model group.

[ example four ] Effect of Eleutherobin D on sugar Water preference of chronically stressed depressed rats

Sugar water preference = sucrose water consumption/total liquid consumption x 100%.

After dosing, 72 h before the sugar bias test, rats were trained to accommodate a 1% aqueous sucrose solution (weight/volume): 2 bottles of 1% sucrose in water were placed in each cage and after 24 h, 1 of these bottles was changed to drinking water for 24 h. After acclimation training was completed, rats were deprived of water and food for 24 h. The sugar water bias test was started at 9:00 am with 1 rat per cage, and the rats had the freedom to choose 2 water bottles (containing 100 mL sucrose solution (1%, weight/volume) and 100 mL drinking water, respectively). After 3 h, the volume of sucrose and drinking water consumed by the rats was recorded. The calculation formula of the sugar water preference degree is as follows: sugar water preference =1% sucrose water consumption volume/(water consumption volume +1% sucrose water consumption volume) × 100%, the results are shown in fig. 4. The results show that the model group has significant difference (p is less than 0.01) with the normal control group, and the sugar water preference degree after 4 weeks of filling the positive drug and eleutheroside D [3.33, 10, 30 mg/(kg.d) ] has significant difference (p is less than 0.01) with the model group.

EXAMPLE V Effect of Eleutherobin D on forced swimming cumulative immobility time in chronically stressed depressed rats

After completion of the administration, forced swim test was performed on rats. The test was performed in two days: rats were allowed to pre-swim in 22 + -2 deg.C deep water for 15 min on day 1, and 24 h later, swimming behavior was observed for 5 min under the same conditions, and the cumulative immobility time was recorded. The determination of the immobility time is based on the fact that the rat is attached to the water surface and only performs a small action of maintaining the balance of the body and exposing the head to the water surface, and the result is shown in FIG. 5. The results show that the model group has significant difference (p < 0.01) from the normal control group, and the cumulative immobility time of forced swimming after 4 weeks of filling positive drug and eleutheroside D [3.33, 10, 30 mg/(kg.d) ] has significant difference (p <0.05, p <0.01 or p < 0.001) from the model group.

EXAMPLE six Effect of Eleutherobin D on neurotransmitter levels in chronically stressed depressed rats

After behavioral assessment, animals were sacrificed and samples of hippocampal, striatal tissue were taken and tested for norepinephrine, 5-hydroxytryptamine, 5-oxindole acetic acid/5-hydroxytryptamine, and dopamine levels. The sample was treated with tissue lysate (0.6 mol/L perchloric acid, 0.5 mmol/L Na)₂EDTA, 0.1 g/L L-cysteine mixed aqueous solution), and then freezing and centrifuging to obtain supernatant; adding perchloric acid precipitant (1.20 mol/L K)₂HPO₄、2.00 mmol/L Na₂Mixed aqueous EDTA solution), and then subjected to refrigerated centrifugation and filtration. The chromatographic conditions and fluorescence detection parameters were as follows: agilent HPLC 1260, Shim-pack C18 column (250 mm. times.4.6 mm, 5 μm) (Shimadzu Japan); the mobile phase is citric acid-sodium acetate buffer (50 mmol/L citric acid, 50 mmol/L sodium acetate, 0.5 mmol/L sodium 1-heptanesulfonate, 5 mmol/L triethylamine, 0.5 mmol/L Na₂EDTA) -methanol (83: 13, v/v) (pH 8.3); the flow rate is 1.0 mL/min; the sample injection amount is 10 mu L; the emission wavelength was 330 nm and the excitation wavelength was 280 nm, the results are shown in FIG. 6. The results showed that the model group was significantly different from the normal control group (p)<0.05 or p<0.01), drench positive medicine, eleutheroside D [3.33, 10, 30 mg/(kg.d)]After 4 weeks the levels of 5-hydroxytryptamine, norepinephrine and 5-oxindole acetic acid/5-hydroxytryptamine and dopamine differed significantly from the model group (p)<0.05 or p<0.01）。

[ example seven ] Effect of Eleutherobin D on Hippocampus neurotrophic factor expression in chronically stressed depressed rats

After behavioral assessment, animals were sacrificed and hippocampal tissue samples were taken and tested for expression levels of hippocampal nerve growth factor, brain-derived neurotrophic factor and glial cell-derived neurotrophic factor. Extracting total RNA from the sample by using an RNA separation kit, and detecting the RNA content by using Nanodrop. A1 mu gRNA sample is taken, is subjected to reverse transcription by a reverse transcription kit to form cDNA, and a real-time quantitative PCR test is carried out by adopting an SYBR Green I chimeric fluorescence method, and the result is shown in figure 7. The results show that the model group has significant difference (p <0.05 or p < 0.01) with the normal control group, and the expression level of the neurotrophic factor after 4 weeks of positive drug administration and eleutheroside D [3.33, 10, 30 mg/(kg.d) ] has significant difference (p <0.05 or p < 0.01) with the model group.

EXAMPLE VIII Effect of Eleutherobin D on the expression of Hippocampus neurotrophic factor receptor in chronically stressed depressed rats

After behavioral assessment, animals were sacrificed and hippocampal tissue samples were taken and tested for expression levels of hippocampal neurotrophic factor receptors (Trk a, B and C). Extracting total RNA from the sample by using an RNA separation kit, and detecting the RNA content by using Nanodrop. A1 mu gRNA sample is taken, is subjected to reverse transcription by a reverse transcription kit to form cDNA, and a real-time quantitative PCR test is carried out by adopting an SYBR Green I chimeric fluorescence method, and the result is shown in figure 8. The results show that the model group has significant difference (p < 0.05) with the normal control group, and the expression level of the neurotrophic factor receptor after 4 weeks of filling the positive drug and eleutheroside D [3.33, 10, 30 mg/(kg.d) ] has significant difference (p <0.05, p <0.01 or p < 0.001) with the model group.

EXAMPLE nine acute toxicity test of eleutheroside D

The experiment was performed according to the maximum tolerated dose method, with 20 ICR mice randomized into 2 groups: blank control group, eleutheroside D test group, each group has 10 animals and females. According to the acute toxicity grading standard in food safety evaluation, the mice before the test are fasted for 12 h, and are intragastrically administered at a dose of 0.4 mL/mouse, and are intragastrically administered for 2 times within 24 h, the daily dose is 15 g, and the mice in a blank control group are intragastrically administered with the same dose of physiological saline. After administration, mice were observed for 72 h mortality, and for food intake and weight change within 7 days in the future; 7 days later, the mouse was cervical spondylopathy freeAfter death and dissection, the pathological changes of the major organs were observed. After the test, the mouse is found to have no poisoning and death after gastric lavage, and in the observation of the next 7 days, no mouse dies, the appearance is healthy, the fur is smooth, the breath and the stool and the urine are normal, no abnormal secretion is found in the nose, the eyes and the oral cavity, and the mental state is good. Compared with the normal control group mice, the method has no obvious difference. After weighing for 7 days, the dissections were sacrificed one by one, and the heart, liver, spleen, lung, kidney, stomach, intestine, and thoracic and abdominal cavity were observed without abnormality in each organ. According to the national standard regulation of the people's republic of China: when the dose of the test substance in the test mouse reaches 15 g/kg, the animal still can not be killed, so that the half lethal dose LD of the test substance is confirmed without accurately measuring the half lethal dose₅₀>15 g/kg. According to the national acute toxicity classification standard, the eleutheroside D belongs to a nontoxic grade.

[ example ten ] eleutheroside D Capsule

100 g of eleutheroside D, 80 g of starch, 24 g of sodium carboxymethyl starch, 17 g of dextrin and a proper amount of 70% ethanol, and preparing 1000 capsules (each capsule contains 100 mg of eleutheroside D) according to a conventional method.

[ EXAMPLE eleven ] Eleutherobin D Capsule

The acanthopanax senticosus glycoside D15 g, the starch 10 g, the sodium carboxymethyl starch 4 g, the dextrin 12 g and a proper amount of 70% ethanol are prepared into 1000 capsules (each capsule contains the acanthopanax senticosus glycoside D15 mg) according to a conventional method.

[ EXAMPLE twelve ] eleutheroside D tablet

Eleutheroside D20 g, lactose 6 g, compressible starch 3 g, hydroxypropyl cellulose 3 g, magnesium stearate 1 g, and 70% ethanol in appropriate amount, and making into 1000 tablets (each tablet contains eleutheroside D20 mg) by conventional tabletting method.

[ EXAMPLE thirteen ] Eleutherobin D Capsule

The acanthopanax senticosus glycoside D20 g, the starch 13 g, the sodium carboxymethyl starch 5 g, the dextrin 5 g and a proper amount of 70% ethanol are prepared into 1000 capsules (each capsule contains the acanthopanax senticosus glycoside D20 mg) according to a conventional method.

[ example fourteen ] Acanthopanax Senticosus glycoside D Capsule

The acanthopanax senticosus glycoside D40 g, the starch 17 g, the sodium carboxymethyl starch 17 g, the dextrin 5 g and a proper amount of 70% ethanol are prepared into 1000 capsules (each capsule contains the acanthopanax senticosus glycoside D40 mg) according to a conventional method.

[ EXAMPLE fifteen ] Acanthopanax Senticosus glycoside D tablet

150 g of eleutheroside D, 55 g of lactose, 15 g of compressible starch, 10 g of hydroxypropyl cellulose, 5 g of magnesium stearate and a proper amount of 70% ethanol, and the eleutheroside D is prepared into 1000 tablets (each tablet contains 150 mg of eleutheroside D) according to a conventional tabletting method.

[ example sixteen ] Eleutherobin D granules

Eleutheroside D6 g, lactose powder 800 g, magnesium stearate 194 g, and 70% ethanol in appropriate amount, and making into granule 1000 g (each granule contains eleutheroside D6 mg) according to conventional preparation method of granule.

[ example seventeen ] Eleutherobin D Soft Capsule

The preparation method comprises the following steps of preparing 1000 granules (each granule contains eleutheroside D20 mg) by using eleutheroside D20 g, soybean oil 10 g, gelatin 5 g, glycerol 5 g and distilled water 7 g according to a conventional soft capsule preparation method.

[ example eighteen ] Eleutherobin D cream

Eleutheroside D0.5 g, stearic acid 100 g, cetyl alcohol 20 g, glycerin monostearate 10 g, liquid paraffin 10 g, methyl hydroxybenzoate 0.8 g, butyl hydroxybenzoate 0.2 g, glycerol 140 g, potassium hydroxide 5 g, ethanol 10 g, and distilled water up to 1000 g.

[ example nineteen ] eleutheroside D injection

Dissolving eleutheroside D25.0 g in 1000 mL of water for injection, stirring thoroughly to dissolve, adding water for injection to 2000 mL, adding activated carbon for injection 2.5 g, heating to 60 deg.C, stirring for 30 min, filtering with carbon rod, filtering the filtrate with 0.25 μm microporous membrane for sterilization, subpackaging in 1000 vials, measuring 2.0 mL/vial, sealing, and sterilizing.

Claims

1. Application of eleutheroside D as the only active component in preparing antidepressant drug is provided.

2. The use according to claim 1, wherein eleutheroside D is used for up-regulating neurotransmitter levels, neurotrophic factors and their receptor expression.

3. The use of claim 1, wherein the eleutheroside D is combined with any pharmaceutically acceptable adjuvant or pharmaceutical excipient to make any pharmaceutically acceptable pharmaceutical dosage form.

4. The use as claimed in claim 3, wherein the dosage form comprises granules, tablets, electuary, soft capsules, dripping pills, ointments or injections.

5. The use according to claim 3, wherein the medicament comprises eleutheroside D in an amount of 0.1-1200 mg.