CN109464433B

CN109464433B - Application of kaurenoic acid in preparation of anti-depression drugs

Info

Publication number: CN109464433B
Application number: CN201811383035.2A
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: 2021-04-23
Anticipated expiration: 2038-11-20
Also published as: CN109464433A

Abstract

The invention discloses an application of kaurenoic acid in preparation of an anti-depression drug. Kaurenoic acid has a remarkable effect of treating depression. The invention relates to an oral administration, a transdermal administration or an injection administration which are prepared from kaurenoic acid and used for treating depression.

Description

Application of kaurenoic acid in preparation of anti-depression drugs

Technical Field

The invention belongs to the technical field of medicines, relates to medical application of kaurenoic acid, and particularly relates to application of kaurenoic acid in medicines 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.

Kaurenoic acid (Kaurenoic acid) is a diterpenoid compound mainly distributed in plants of the genus Araliaceae. However, the main effective components of acanthopanax are lignans and glycosides thereof, triterpenes and glycosides thereof, coumarins, flavones and the like, and the kaurenoic acid is low in content in acanthopanax. The kaurenoic acid is white powder, and has a molecular formula: c₂₀H₃₀O₂Molecular weight: 302.46 has effects in relieving vascular smooth muscle, resisting spasm, resisting platelet aggregation, resisting tumor, lowering blood pressure, promoting urination, and relieving inflammation. At present, the application of kaurenoic acid in preparing anti-depression drugs is not available.

Disclosure of Invention

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

the invention relates to an application of kaurenoic acid in depression resistance, which can remarkably regulate the level of neurotransmitters and 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 kaurenoic acid can be applied to preparation of anti-depression drugs, and the drugs have the application of treating depression.

Kaurenoic acid can be obtained commercially or prepared by prior art methods.

The kaurenoic acid 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 kaurenoic acid to be administered in the present invention varies depending on the condition, body weight, administration method, and the like of the patient. For example, the dosage is 0.1-200 mg/(kg.d) for non-oral administration, intravenous administration and intestinal administration, and 0.4-800 mg/(kg.d) for oral administration.

The kaurenoic acid provided by the invention is used for performing an intragastric lavage test on mice by adopting a maximum tolerance method, and the result shows that the mice are healthy and do not have abnormal reaction after the effect, the anatomy does not have obvious pathological changes, the organ index and the serum index do not have obvious difference from the normal group, and the kaurenoic acid has no obvious difference from the normal group to the oral LD of the mice₅₀>15 g/kg, which shows that the kaurenoic acid has no toxic effect on mice and is safe.

Compared with the prior art, the invention has the beneficial effects that: kaurenoic acid can remarkably shorten forced swimming and tail suspension accumulation immobility time of normal mice, has a remarkable anti-depression effect on chronic stress depressed rats, can remarkably improve the 5-hydroxytryptamine, norepinephrine, 5-oxindole acetic acid/5-hydroxytryptamine level and striatal dopamine level of depressed rats in the hippocampal region, remarkably up-regulates the neurotrophins and receptor expression of depressed rats in the hippocampal region, and acute toxicity experiments show that kaurenoic acid is non-toxic. Therefore, the kaurenoic acid can be applied to preparation of anti-depression drugs. The invention provides a new clinical application of kaurenoic acid and enlarges the application range of kaurenoic acid.

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 kaurenoic acid reference substance is prepared by self, has the purity of more than or equal to 98 percent, and is dissolved in water to prepare the administration concentration (0.75, 1.5, 3 mg/mL or 5, 10, 20 mg/mL).

[ example one ] Effect of kaurenoic acid 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, a kaurenoic acid group [7.5, 15, 30 mg/(kg.d) ], an 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, and were gavaged 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 mouse after the positive drug and the kaurenoic acid [7.5, 15, 30 mg/(kg.d) ] are obviously different from the model group (p <0.05 or p < 0.01).

[ example two ] Effect of kaurenoic acid on cumulative immobility time in mice tail suspension

ICR mice, male, were bred for 7 days to adapt to the new environment. Mice were divided into a blank control group, a kaurenoic acid group [7.5, 15, 30 mg/(kg.d) ], an 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, and were gavaged 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, kaurenoic acid [7.5, 15, 30 mg/(kg.d) ], levodopa and galantamine act is significantly different from that of the model group (p is less than 0.05 or p is less than 0.01).

[ example III ] Effect of kaurenoic acid on body weight in chronically stress-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, a kaurenoic acid group [5, 10, 20 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 of 12 rats, and were 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 body weight has significant difference (p < 0.05) with the model group after 4 weeks of filling the positive drug and the kaurenoic acid [5, 10, 20 mg/(kg.d) ].

[ example four ] Effect of kaurenoic acid on sugar preference in chronically stress-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 < 0.01) with the normal control group, and the sugar water preference degree after 4 weeks of filling the positive drug and the kaurenoic acid [5, 10, 20 mg/(kg.d) ] has significant difference (p < 0.01) with the model group.

EXAMPLE five Effect of kaurenoic acid on cumulative immobility time in forced swimming of 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 accumulative immobility time of forced swimming after 4 weeks of filling positive drug and kaurenoic acid [5, 10, 20 mg/(kg.d) ] has significant difference (p <0.05, p <0.01 or p < 0.001) from the model group.

EXAMPLE sixty effects of kaurenoic acid 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. Result display modelThe group has significant difference (p) from the normal control group<0.05 or p<0.01), drench positive medicine, kaurenoic acid [5, 10, 20 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 kaurenoic acid on the expression of neurotrophic factor in Hippocampus of 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 kaurenoic acid [5, 10, 20 mg/(kg.d) ] has significant difference (p <0.05 or p < 0.01) with the model group.

EXAMPLE eigh Effect of kaurenoic acid on Hippocampus neurotrophic factor receptor 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 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 the kaurenoic acid [5, 10, 20 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 kaurenoic acid

The experiment was performed according to the maximum tolerated dose method, with 20 ICR mice randomized into 2 groups: blank controlGroups and kaurenoic acid test groups, 10 of the groups each have half of male and female. 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; after 7 days, the mice were sacrificed by cervical dislocation and the pathological changes of the major organs were observed after dissection. 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, kaurenoic acid belongs to a non-toxic grade.

[ example ten ] kaurenoic acid capsules

100 g of kaurenoic acid, 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 kaurenoic acid) according to a conventional method.

[ EXAMPLE undecenoic acid Capsule

The preparation method comprises the following steps of preparing 1000 capsules (each capsule contains 15 mg of kaurenoic acid) by using 15 g of kaurenoic acid, 10 g of starch, 4 g of sodium carboxymethyl starch, 12 g of dextrin and a proper amount of 70% ethanol according to a conventional method.

[ example twelve ] kaurenoic acid tablet

20 g of kaurenoic acid, 6 g of lactose, 3 g of compressible starch, 3 g of hydroxypropyl cellulose, 1 g of magnesium stearate and a proper amount of 70% ethanol, and the kaurenoic acid tablet is prepared into 1000 tablets (each tablet contains 20 mg of kaurenoic acid) by a conventional tabletting method.

[ EXAMPLE thirteen ] kaurenoic acid capsules

20 g of kaurenoic acid, 13 g of starch, 5 g of sodium carboxymethyl starch, 5 g of dextrin and a proper amount of 70% ethanol, and preparing 1000 capsules (each capsule contains 20 mg of kaurenoic acid) according to a conventional method.

[ example fourteen ] kaurenoic acid capsules

40 g of kaurenoic acid, 17 g of starch, 17 g of sodium carboxymethyl starch, 5 g of dextrin and a proper amount of 70% ethanol, and preparing 1000 capsules (each capsule contains 40 mg of kaurenoic acid) according to a conventional method.

[ example fifteen ] kaurenoic acid tablet

150 g of kaurenoic acid, 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 kaurenoic acid tablet is prepared into 1000 tablets (each tablet contains 150 mg of kaurenoic acid) by a conventional tabletting method.

[ example sixteen ] kaurenoic acid granules

6 g of kaurenoic acid, 800 g of lactose powder, 194 g of magnesium stearate and a proper amount of 70% ethanol, and the granules are prepared into 1000 g of granules (each g of granules contains 6 mg of kaurenoic acid) according to a conventional preparation method of the granules.

[ example seventeen ] kaurenoic acid Soft Capsule

The preparation method comprises the following steps of preparing 1000 capsules (each capsule contains 20 mg of kaurenoic acid) according to a conventional soft capsule preparation method by using 20 g of kaurenoic acid, 10 g of soybean oil, 5 g of gelatin, 5 g of glycerol and 7 g of distilled water.

[ example eighteen ] kaurene yogurt cream

0.5 g of kaurenoic acid, 100 g of stearic acid, 20 g of cetyl alcohol, 10 g of glycerol monostearate, 10 g of liquid paraffin, 0.8 g of methylparaben, 0.2 g of butylparaben, 140 g of glycerol, 5 g of potassium hydroxide, 10 g of ethanol and distilled water added to 1000 g.

[ example nineteen ] kaurenoic acid injection

Dissolving kaurenoic acid 25.0 g in 1000 mL of water for injection, fully stirring for dissolving, adding water for injection to 2000 mL, adding activated carbon for injection 2.5 g, heating to 60 ℃, stirring for 30 min, filtering with a carbon rod, filtering the filtrate with a 0.25 mu m microporous filter membrane for sterilization, subpackaging in 1000 penicillin bottles with the content of 2.0 mL/bottle, sealing, and sterilizing to obtain the kaurenoic acid injection.

Claims

1. Application of kaurenoic acid in preparation of anti-depression drugs.

2. The use according to claim 1, wherein kaurenoic acid is useful in up-regulating neurotransmitter levels, neurotrophic factors, and receptor expression.

3. The use of claim 1, wherein the kaurenoic acid is formulated with any pharmaceutically acceptable excipient or pharmaceutical vehicle into any pharmaceutically acceptable pharmaceutical dosage form.

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

5. The use according to claim 3, wherein the medicament comprises 0.1 to 800mg of kaurenoic acid.