WO2024131267A1

WO2024131267A1 - Electronic cigarette atomization liquid having antidepressant effect, and preparation method therefor

Info

Publication number: WO2024131267A1
Application number: PCT/CN2023/126570
Authority: WO
Inventors: 林光榕; 叶华; 吴铁; 吴科峰; 戚怡; 顾娜
Original assignee: 惠州市新泓威科技有限公司
Priority date: 2022-12-19
Filing date: 2023-10-25
Publication date: 2024-06-27
Also published as: CN116158550A

Abstract

An electronic cigarette atomization liquid having an antidepressant effect, prepared from the following components in parts by weight: 0.1-4 parts of limonene, 0.1-5 parts of nicotine, 1-8 parts of menthol, 70-80 parts of propylene glycol, and 10-30 parts of glycerol. The electronic cigarette atomization liquid having the antidepressant effect overcomes the defects that existing electronic cigarette atomization liquids containing nicotine on the market do not have any antidepressant effect and depressive symptom alleviation effect; additionally, because limonene has an anti-inflammatory effect on lung inflammation, the inflammation and damage of lung, trachea, and bronchus caused by long-term smoking of smokers can be alleviated. In addition, a preparation method for the electronic cigarette atomization liquid having the antidepressant effect is provided.

Description

Electronic cigarette aerosol liquid with antidepressant effect and preparation method thereof

Technical Field

The invention belongs to the technical field of electronic cigarette atomizer liquid preparation, and particularly relates to an electronic cigarette atomizer liquid with antidepressant effect and a preparation method thereof.

Background technique

Depression is a common and widespread mental illness worldwide, which has a significant impact on the quality of human life. Studies have shown that depression is the result of the combined effects of psychological, physiological, and social factors. Many factors may be the cause of depression, including genetics, external adverse stimuli or internal conflicts, smoking, alcohol and drug abuse, chronic diseases, and improper diet. According to the incidence, it can be divided into mild depression and severe depression, and severe depression poses a serious threat to human health. With the development of society, the increase in stress and the effects of various other factors have caused the incidence of depression to increase year by year. The World Health Organization ranks major depression as the third largest cause of disease burden in the world, and predicts that by 2030, the disease will become the first cause of disease burden. More than 350 million people worldwide are affected by major depression, which is characterized by significant changes in mood (persistent depression or lack of pleasure), accompanied by obvious psychological and nutritional changes, such as drowsiness, anorexia, subjective fatigue, loss of motivation, guilt and despair of conscience, difficulty maintaining concentration, suicidal thoughts and behaviors. Globally, the burden of depression is increasing, especially among women.

At present, the treatment of depression is mainly with antidepressants, and the treatment goal is symptom relief and functional recovery. However, experimental and clinical evidence shows that the efficacy of antidepressants is not ideal. In fact, about 30-40% of patients are completely relieved after one course of treatment, while 30% of patients still have residual symptoms, increased risk of relapse, and affect social function after clinically significant relief. At present, the treatment of depression mainly relies on chemical synthetic drugs. However, this method generally has the disadvantages of narrow antidepressant spectrum, slow onset, and large side effects. Compared with chemical synthetic drugs, natural drugs have unique advantages in the prevention and treatment of depression. It is reported that these many volatile oils containing limonene can easily pass through the blood-brain barrier into the brain, have good antidepressant effects, and have few toxic side effects.

The relationship between monoamine oxidase activity and neurotransmitter levels has been shown to be associated with neurological and psychiatric diseases, and monoamine oxidase has become a valuable therapeutic target in neuropharmacology. Monoamine oxidase-A inhibitors are currently used to treat anxiety and depression. Since the 1950s, the first monoamine oxidase inhibitor, epronil, has been used as an antidepressant treatment because the levels of the three major monoamine substances (such as norepinephrine, dopamine and serotonin) are regulated in the brain. The monoamine transmitter imbalance hypothesis is a widely accepted pathogenesis of depression. In patients with depression, the reduction of norepinephrine and 5-hydroxytryptamine levels in the brain and the induction of MAO-A expression are the most important pathogenic factors of depression. In addition, depression is associated with monoamine oxidase-A functional polymorphism genes and 5-hydroxytryptamine gene signaling pathways. In the case of depression, the level of monoamine oxidase A in the brain increases. Therefore, traditional antidepressants inhibit monoamine oxidase A to reduce the activity of the 5-HT signaling pathway and exert a therapeutic effect. Inhibitors of monoamine oxidase-A have been found to have antidepressant properties and have been used in the treatment of depression for more than 50 years. Since monoamine oxidase-A mainly metabolizes 5-HT and NE, inhibitors of this enzyme are mainly used to treat depression. Studies have shown that monoamine oxidase inhibitors inhibit the deamination or metabolism of neurotransmitters. Monoamine oxidase inhibitors are also determined by their selectivity: some inhibitors selectively inhibit MAO-A (such as moclobemide), some selectively inhibit MAO-B (such as pargyline and selegiline), and some are non-selective (such as phenelzine and tronicyclopropane) and can inhibit both MAO-A and MAO-B. Early monoamine oxidase inhibitors inhibited monoamine oxidase irreversibly, and when they interacted with monoamine oxidase, they permanently inactivated the enzyme. Later developed monoamine oxidase inhibitors, such as moclobemide, are reversible, meaning that when the inhibitor is separated from the enzyme, MAO enzyme activity can be restored again. Although MAO-A inhibitors are a therapeutic mechanism for treating mental illness, some adverse reactions may occur when MAO-A inhibitors are taken in excess or in combination with other drugs, dietary supplements, or botanicals, such as hepatotoxicity, sedation, cognitive impairment, movement disorders, aggression, sexual dysfunction, drug tolerance, and dependence. At the same time, excessive MAO-A inhibition in the liver can hinder the metabolism of tyramine in the diet, ultimately leading to a hypertensive crisis. Therefore, it is necessary to find natural, non-toxic inhibitors that can replace traditional MAO-A inhibitors.

Limonene is a monoterpene from the Rubiaceae family that has biological properties such as antioxidant, anti-inflammatory, anticancer, antinociceptive, and gastroprotective properties. Recently, there has been a great interest in studying the pharmacological effects of limonene in various chronic diseases due to its mitigating effects on oxidative stress and inflammation and its role in regulating apoptotic cell death. Available antidepressants are designed to target and restore monoamine imbalances. Studies have found that current antidepressants are associated with severe adverse reactions and that their effects vary from individual to individual. Therefore, the discovery of new drugs with the ability to normalize mood is of vital importance. Monoterpenes have been found to have mood-improving and mood-improving effects by acting directly on the central nervous system and olfactory nerves. Citrus essential oil (OEO), which contains limonene as its main component, has been observed to have an antidepressant effect in a study of a chronic unpredictable mild stress model created in mice. The researchers found that the brains of treated mice had higher levels of limonene than other compounds. They observed that OEO inhalation significantly improved the depressive-like behavior of the model mice, with reduced weight loss, sucrose preference, curiosity, and activity, a shorter immobility time, and a reduction in dyslipidemia. Limonene is the most abundant compound in the brain of mice sniffing OEO environment and after sniffing, and it is not immediately metabolized in the brain. In addition, inhalation of limonene can significantly restore the depressive behavior induced by the model, the hyperactivity of the hypothalamic-pituitary-adrenal axis, and the reduction of monoamine neurotransmitter levels, downregulating the expression of brain-derived neurotrophic factor and its receptors in the hippocampus. Therefore, the researchers believe that the improvement of neuroendocrine, neurotrophic and monoaminergic systems is related to the antidepressant effect of limonene. [Zhang LL, Yang ZY, Fan G., Ren JN, Yin KJ, Pan SY Antidepressant-like Effect of Citrus sinensis (L.) Osbeck Essential Oil and Its Main Component Limonene on Mice.J Agric Food Chem. 2019 Dec 18;67(50):13817-13828.]. Other researchers have demonstrated that oral D-limonene can reduce the mechanical hyperalgesia induced by preserved nerve injury, showing anti-hyperalgesia and antidepressant effects. Inhaled EOs may modulate brain health and function related to mood and neurodegeneration, reflecting their bioavailability in the brain. [Piccinelli AC, Antihyperalgesic and antidepressive actions of (R)-(+)-limonene, α-phellandrene, and essential oil from Schinus terebinthifolius fruits in a neuropathic pain model. Nutr Neurosci. 2015 Jul;18(5):217-24]. Other studies have demonstrated the ability of limonene to increase GABA levels in the rat brain, with an anxiolytic effect, and others have demonstrated neuroprotective effects in neurodegenerative diseases, including Alzheimer's disease, multiple sclerosis, epilepsy, anxiety, and stroke. We note the effects of limonene on the respiratory tract, particularly acute lung injury, a serious disease associated with inflammation, high morbidity and mortality, and no specific drugs available for treatment. Studies have found that limonene can prevent acute lung injury caused by intranasal LPS in mice. Limonene shows effective lung protection. The researchers believe that the effect of limonene is caused by improving lung function through its anti-inflammatory activity. Limonene is able to reduce inflammatory neutrophil infiltration and subsequent myeloperoxidase activity (MPO). Inflammatory cytokines such as TNF-α, IL-1β, and IL-6 are considered therapeutic targets for acute lung injury; limonene treatment reduces the activity of these cytokines. Limonene is able to attenuate the activation of NF-κB, ERK, JNK, and p38 MAPK signaling pathways, showing anti-inflammatory effects in preventing LPS-induced acute lung injury and demonstrating potential new biological activities for limonene. Bronchial asthma is a chronic inflammatory disease that affects 300 million people worldwide. The clinical characteristics of this disease are allergen-induced airway obstruction, chronic eosinophilic airway inflammation, excessive mucus secretion, airway remodeling, and nonspecific airway hyperresponsiveness. Due to these airway abnormalities, individuals susceptible to asthma or allergic airway disease are believed to be more sensitive to inhaled irritants. A study was conducted to evaluate whether limonene could reduce allergic airways and improve asthma symptoms in a dust mite-induced (DER) airway inflammation model. The results obtained showed that oral treatment with limonene significantly reduced immunoglobulin E (IgE), an important marker of allergic inflammation, representative Th-2 cell types, responsible for the production of proinflammatory cytokines, the chemotactic cytokine MCP-1, important for the influx of inflammatory cells, and TGF-β1, which is associated with the development of asthmatic airway remodeling. Histopathological changes also showed that limonene treatment effectively reduced the influx of eosinophils into the lungs, airway fibrosis, and alleviated collagen deposition. In addition, limonene treatment was able to alleviate bronchoconstriction induced by the administration of acetylcholine in DER mice. These results suggest that limonene has an effective therapeutic effect on allergic airways and asthma.

Mint is the above-ground part of the herbaceous plant mint of the Lamiaceae family. It is pungent and cool, and belongs to the lung and liver meridians. It has the effects of dispersing wind-heat, clearing heat and relieving sore throat, dispelling wind and rash, and soothing the liver and relieving depression. Among them, the aromatic compounds of mint, such as L-menthol, have the effects of aromatic awakening and refreshing the mind. The content of L-menthol in mint oil can be as high as 87%. The aromatic smell of L-menthol can act on the brain nervous system. Literature reports that L-menthol can significantly inhibit the expression of glutamate receptor 1 and acetylcholinesterase in the hippocampus of rats, thereby improving the cognitive ability of rats and improving memory. Experimental results show that the aroma of L-menthol can enhance the short-term memory of cockroaches and is related to the storage and retrieval of thinking information. L-menthol can enhance the bioavailability of nerve growth factor in the hippocampus of rats. L-menthol acts on hippocampal neurons, which can delay the desensitization of γ-aminobutyric acid receptors and selectively enhance the affinity of hippocampal neurons in rats. L-menthol can control the autonomic nervous system and also has an effect on low mood and depression symptoms. The interaction between menthol and nACh receptors has been investigated in several early studies. Nicotine is a major irritant contained in tobacco smoke, causing burning or stinging sensations in the oral or nasal mucosa [9]. Nicotine-induced sensations are thought to involve activation of nACh receptors, including those composed of the α7 subunit, which are expressed in sensory fibers innervating these tissues as well as in the bronchial and tracheal epithelial cells of lung tissue. Menthol may reduce nicotine-induced irritation and sensory perception. More recently, menthol has been shown to act as a counterirritant to inhaled cigarette smoke, suggesting that menthol may reduce nicotine-induced responses. In addition to sensory responses, one of the major physiological effects of nicotine is a decrease in body temperature due to cutaneous vasodilation, an effect that originates in the brain and may be mediated by hypothalamic nicotinic receptors. Both chronic and acute menthol administration reduce the effects of nicotine on body temperature. Studies have shown that the traditional Chinese medicine compound Xiaoyaosan containing mint has significant therapeutic effects on depression, with mint and bupleurum being considered the main antidepressant drugs in the formula. There is limited data on the use of mint in the treatment of anxiety and depression. The European Medicines Agency has not included these indications in its herbal monographs and has approved peppermint for the following indications: mild gastrointestinal spasms, flatulence, abdominal pain, indigestion, mild headaches, coughs and colds, localized muscle pain, and itching of intact skin. The scent of peppermint has been reported to reduce fatigue and improve mood and sleep. There are also reports that peppermint infusions and fresh parts have a positive effect on the mental health of college students. Students in the treatment group reported better memory function and sleep quality, and reduced anxiety, but this study was not blinded. A recent study reported the beneficial effects of 7 days of peppermint oil aromatherapy on sleep quality scores in cancer patients.

The zebrafish (Danio rerio) is a vertebrate model that is widely used in biomedical research, including neurotoxicology studies. Zebrafish exhibit an overall nervous system and neurotransmitter systems similar to humans, including glutamatergic, cholinergic, serotonergic, dopaminergic, adrenergic, GABA, and histamine. The mature zebrafish central nervous system (CNS) is well developed and more complex and can be used to model complex brain disorders, including anxiety and depression. There are several experimental assays that can measure behavioral learning changes in adult zebrafish, such as the rotational escape test, bite test, novel tank test, place preference test, T-maze, plus-maze, and Y-maze assays. Most of the tools used to assess learning and memory in animal models involve visual stimulation, including color preference. Zebrafish can discriminate colors and exhibit spontaneous approach or avoidance behaviors. Some studies support color-based learning and memory paradigms or experiments involving aversion, anxiety, or fear in zebrafish. Zebrafish prefer blue and green colors and avoid yellow and red. The visual system of zebrafish includes a retina with cone cells that are sensitive to red, green, blue, and ultraviolet light; in addition, zebrafish are diurnal animals, which makes them an ideal model for the study of developing cognitive responses to visual cues. In addition, reserpine has the effect of lowering blood pressure and slowing heart rate, and was originally a good antihypertensive and sedative. However, previous studies have shown that acute reserpine application can cause depression.

Electronic cigarettes are mainly used to replace tobacco cigarettes. Electronic cigarettes can heat and evaporate the stored electronic cigarette atomizer liquid or electronic cigarette oil into aerosol for users to inhale. Currently, the nicotine-containing electronic cigarettes on the market do not contain effective antidepressant ingredients because their atomizer liquid does not contain antidepressant effects. Therefore, existing electronic cigarettes have no antidepressant effect or have no effect on improving depressive symptoms.

technical problem

The purpose of the present invention is to provide an electronic cigarette atomizer liquid with antidepressant effect and a preparation method thereof. After a large number of experimental drug combinations and experimental observations, we found that in the electronic cigarette atomizer liquid containing nicotine, the electronic cigarette atomizer liquid with limonene and menthol as main additives can inhibit the activity of monoamine oxidase and play an antidepressant effect. This nicotine atomizer liquid containing limonene and menthol has an antidepressant effect and can replace the effect of monoamine oxidase A inhibitors for people suffering from mild depression and anxiety, thereby alleviating their symptoms.

Technical Solutions

The technical solution of the present invention is an electronic cigarette atomizer liquid with antidepressant effect, and its preparation components include: 0.1-4 parts of limonene, 0.1-5 parts of nicotine, 1-8 parts of menthol, 70-80 parts of propylene glycol, and 10-30 parts of glycerol in parts by weight.

Preferably, the limonene is 0.5 parts, the nicotine is 4 parts, the menthol is 3 parts, the propylene glycol is 72.5 parts, and the glycerol is 20 parts.

Another technical solution of the present invention is a method for preparing an electronic cigarette atomizer liquid having an antidepressant effect, comprising the following steps:

S1. Weigh each component according to the weight ratio of the formulated components;

S2. The components are sequentially added to the centrifuge tube, stirred and mixed at a stirring rate of 200 rad / min;

S3. Soaking at 40°C for 480 minutes;

S4. Heat to 60°C, keep for 20 minutes and then cool to room temperature;

S5. Filter out impurities and you are done.

Beneficial Effects

The electronic cigarette atomizer liquid with anti-depression as the main function prepared by using menthol and limonene as additives solves the defects that the electronic cigarette atomizer liquid containing nicotine currently on the market has no anti-depressant effect and no improvement effect on depressive symptoms. At the same time, since limonene has an anti-inflammatory effect on lung inflammation, it can reduce the inflammation and damage of the lungs, trachea and bronchus caused by long-term smoking in smokers; at the same time, since the electronic cigarette atomizer liquid composed of limonene and menthol of the present invention has the effect of relieving anxiety, tension, irritability and the like to a certain extent after being absorbed by users through the atomization of electronic cigarettes, it can also increase memory, improve thinking, and have the effect of anti-depression, and also has a certain protective effect on lung damage.

Best Mode for Carrying Out the Invention

The present invention uses zebrafish as a model to establish a depression model using reserpine, observes the antidepressant effect of an electronic cigarette atomizer containing nicotine and menthol and limonene as main additives, and finds that the electronic cigarette atomizer has a very obvious antidepressant effect and can be used as an electronic cigarette atomizer with antidepressant effect.

The invention discloses an electronic cigarette atomizer liquid with antidepressant effect, wherein the components thereof include, by weight: 0.1 to 4 parts of limonene, 0.1 to 5 parts of nicotine, 1 to 8 parts of menthol, 70 to 80 parts of propylene glycol, and 10 to 30 parts of glycerol.

Embodiments of the present invention

The present invention will be described in detail below through specific embodiments.

Example

An electronic cigarette atomizer liquid with antidepressant effect, wherein the prepared components include, by weight, 0.5 parts of limonene, 4 parts of nicotine, 3 parts of menthol, 72.5 parts of propylene glycol, and 20 parts of glycerol.

The method for preparing the electronic cigarette atomizer liquid having an antidepressant effect comprises the following steps:

S1. Weigh each component according to the weight ratio of each component;

S2. Add each component to the centrifuge tube in sequence, stir and mix thoroughly at a stirring rate of 200 rad/min;

S3. Soaking at 40°C for 480 minutes;

S4. Heat to 60°C, keep for 20 minutes and then cool to room temperature;

S5. Filter out impurities and you are done.

The formula of the electronic cigarette atomizer liquid with antidepressant effect of the present invention (hereinafter referred to as "limonene nicotine atomizer liquid") is verified by a zebrafish model. The main experimental methods and results are as follows:

Evaluation of the antidepressant efficacy of experimental limonene nicotine atomizer

1. Test on safe concentration of limonene nicotine atomizing liquid

1 Materials and methods

1.1 Experimental organisms

The zebrafish broodstock used in the experiment were the AB strain introduced from the National Zebrafish Resource Center and were raised in a 28.5°C constant temperature water bath and a 14/10h light/dark cycle circulating water culture system. In this experiment, 6hpf embryos were selected as the fish for sample safety concentration testing.

1.2 Test substances

1.3 Instruments and Equipment

Dissecting microscope, zebrafish culture system (Shanghai Haisheng Biological Experiment Equipment Co., Ltd.), 24-well plate, various specifications of pipette tips, Pasteur pipettes, etc., constant temperature incubator.

1.4 Experimental methods

1.4.1 Collection of zebrafish embryos: Select healthy and sexually mature zebrafish and place them in a glass tank with an incubator at a constant temperature of 28°C at a ratio of 2:3. The females and males are separated by a partition and the glass tank is shaded with a black cloth. On the second day, remove the black cloth, pull out the partition, and illuminate with light. Zebrafish are stimulated by light to lay eggs by females and ejaculate by males. After the spawning is completed, remove the unfertilized embryos and foreign bodies on the fertilized embryos, collect the embryos, and incubate them in a 6-well cell culture plate at a constant temperature of 28°C.

1.4.2 Safety concentration test: The preliminary experiment determines the initial range of lethality of each drug to fry, that is, the two concentration ranges that can make the fry survive and die, to ensure the effectiveness of the lethal concentration. Set 6 concentration gradients of 0%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% according to a certain proportion. Set 3 replicates for each concentration, and 10 embryos for each replicate. Use a bus pipette to randomly select normal fertilized eggs and put them evenly into a 24-well plate with sample solution. The experiment is carried out in a constant temperature box at 28 ℃, and the light cycle is set to 12 h:12 h. The toxicological endpoints during the development process are observed and recorded regularly. Observe and record the mortality rate of the embryos, observe the embryonic development under a dissecting microscope at least twice a day, and judge the eggs that are white to the naked eye or black under an inverted microscope as dead individuals during the observation process, and pick them out from the culture plate in time. The observation should be carried out at room temperature of 28 ℃ as much as possible, and the action should be quick, and the eggs should be put back into the constant temperature box after completion. The toxicological endpoint selected for this study was mortality at 96 hours of embryonic development.

1.5 Statistics

The LC50 was calculated using the Spearmam-Karber method.

2. Experimental results

2.1 Determination of LC50 and safe concentration of limonene nicotine atomized liquid for 96 hours

The 96h experimental results of limonene nicotine atomized liquid and the calculation results of LC50 and safe concentration are shown in Table 1:

[Corrected 05.12.2023 in accordance with Rule 26]
Table 1: Experimental results of 96h limonene nicotine atomized liquid and calculation process of LC50

[Corrected 05.12.2023 in accordance with Rule 26]

According to the experimental results, the LC50 of the 96h acute toxicity test of limonene nicotine aerosol liquid is 0.24.

2.2 Calculation of safe concentration of limonene nicotine atomizing liquid

LC50 refers to the concentration required to cause death in 50% of a group of subjects. According to the 96-hour acute toxicity test of limonene nicotine aerosol solution, the LC50 is 0.24. The results of calculating the safe concentrations LC50/10, LC50/5 and LC50/3 are shown in Table 2.

Table 2 Acute toxicity test and safe concentration of limonene nicotine atomized liquid

Note: LC50: refers to the concentration required to cause death of 50% of a group of subjects.

According to the experimental results, it was concluded that the limonene nicotine atomized liquid could obtain an effective LC50 of 96 hours. At the same time, it was found that the safe concentration of the sample was low. The subsequent efficacy evaluation used 1/10, 1/5, and 1/3 of the LC50 as the test concentration (see Table 2 for details) to further evaluate the efficacy of the limonene atomized liquid.

2. Effect of limonene nicotine atomized liquid on monoamine oxidase activity

1 Materials and methods

1.1 Experimental organisms

The zebrafish broodstock used in the experiment were the AB strain introduced from the National Zebrafish Resource Center. In this chapter, 5 dpf fry were selected as the experimental fish.

1.2 Test substances and main reagents

(1) Reserpine (4 mg/L): (Sigma | Lot: 83580)

Preparation of working solution: Use a 1/10,000 balance to weigh 2 mg of Reserpine, add 100 μl of DMSO, fully dissolve it, then add 500 ml of ultrapure water, the working solution concentration is 4 mg/L, and the DMSO concentration is 0.2‰.

(2)Toloxatone (200 μM): (MCE | Lot: HY-14196)

Preparation of working solution: Use a 1/10,000 balance to weigh 50 mg of Toloxatone, add 965.11 μl of DMSO, concentration 250 mM, dilute 1250 times, working solution concentration 200 μM, DMSO concentration 0.8‰.

(3) Monoamine oxidase (MAO) activity detection kit: (Solarbio | Lot: BC0015);

(4) BCA protein concentration assay kit (Biyuntian | Lot: P0012S)

(5) Preparation of test solution:

The limonene nicotine atomized liquid was prepared according to the formula of Example 1. After preparation, it was divided into three concentrations for experiments:

Low concentration group of limonene nicotine aerosol solution: 23 µl of the test substance was aspirated with a pipette and dissolved in 100 ml of ultrapure water for later use. The working solution concentration was 0.023%.

Limonene nicotine aerosol liquid concentration group: use a pipette to draw 48 µl of the test substance and dissolve it in 100ml of ultrapure water for later use. The working solution concentration is 0.048%.

High concentration group of limonene nicotine aerosol solution: 80µl of the test substance was aspirated with a pipette and dissolved in 100ml of ultrapure water for later use. The working solution concentration was 0.080%.

1.3 Instruments and Equipment

1.4 Experimental methods

1.4.1 Drug treatment and administration design (see Table 3);

Table 3 Drug administration sequence for testing

Note: “+” means that this drug is included; “-” means that this drug is not added;

For example, methylene blue was added to the culture water from 5 hours after fertilization to the 5th day for incubation (methylene blue inhibits the growth of cultured water molds), and methylene blue, Reserpine, limonene nicotine aerosol solution, etc. were added to the culture water from the 5th day to the 6th day for co-administration efficacy evaluation.

1.4.2 Experimental group settings;

Experimental group setting, MAO detection according to the detection needs. Each concentration group was repeated in two groups, 30 fish in each group, a total of 60 fish larvae, the experimental container was a 6-well plate, 10 ml of working solution was added to each well, and the efficacy evaluation experimental group was set up (see Table 4).

Table 4 Experimental group settings for the efficacy evaluation of limonene nicotine atomizer liquid

Note: All larvae in the experimental groups were normal, with no mortality or developmental deformities. However, during the drug administration process, most of the larvae in the experimental groups floated on the water surface and sank with a slight shake. Their activities were normal.

1.4.3 MAO ELISA

Refer to the instructions of the Monoamine Oxidase (MAO) Activity Detection Kit: (Solarbio | Lot: BC0015) and take samples at the end of the test. Each experimental group has 60 fry. Protein is extracted according to the instructions of the kit. Protein quantification is required before MAO testing. Refer to the instructions of the BCA Protein Concentration Determination Kit (Biyuntian | Lot: P0012S). MAO activity is calculated by measuring the rate of decrease in light absorption at 360nm.

1.5 Results and Data Analysis

MAO catalyzes the deamination of monoamine substrates to generate aldehydes, which are further oxidized to generate acids. The substrate has a characteristic absorption peak at 360nm. The rate of decrease in light absorption at 360nm is measured by an enzyme marker to calculate the MAO activity. SPSS was used to process the data and compare the significance of the differences between the experimental groups (* 0.01＜P≤0.05, ** P≤0.01). At least 90% of the fish embryos survived after the exposure of each test group, otherwise the results of the corresponding test group were invalid. The experimental results of MAO activity are shown in Table 5:

Table 5: MAO activity in each group

From the results in Table 5, it can be seen that the reserpine model group can significantly increase the amount of MAO in zebrafish, the increase is 591%, and the positive drug group (Toloxatone) can significantly reduce the MAO content in the model group, the enzyme activity is 86.25, and the enzyme activity is reduced by 13.8%. Compared with the model group, the three concentration groups of the sample can significantly reduce the MAO content in the model group. Compared with the model group, the enzyme activity of the low concentration group is reduced by 35.1%; compared with the model group, the enzyme activity of the medium concentration group is reduced by 72.0%, and the enzyme activity of the high concentration group is reduced by 79.2%. The experimental results suggest that limonene nicotine atomized liquid has a significant inhibitory effect on the increase of zebrafish monoamine oxidase caused by reserpine. Compared with the positive control drug Toloxatone, the three test concentrations have a more obvious inhibitory effect, and this inhibitory effect is related to the concentration of the drug. Limonene nicotine aerosol liquid can significantly reduce the amount of MAO, and the effect becomes more obvious as the concentration increases.

3. Behavioral effects of limonene nicotine atomizer on antidepressant efficacy

1 Materials and methods

1.1 Experimental organisms

1.2 Test substances and main reagents

(1) Reserpine (4 mg/L): (Sigma | Lot: 83580)

(2)Toloxatone (200μM): (MCE | Lot: HY-14196)

Positive drug control; Preparation of working solution: Use a 1/10,000 balance to weigh 50 mg of Toloxatone, add DMSO to a volume of 965.11 μl, a concentration of 250 mM, dilute 1250 times, working concentration 200 μM, DMSO concentration 0.8‰.

(3) Preparation of test substances:

1.3 Instruments and Equipment

Main instruments and equipment are shown in the table below:

1.4 Experimental methods

At the same time as the MAO test, at the end of the experiment, behavioral monitoring was performed on the live larvae that had been administered for 24 hours, and the experimental group settings were consistent with the experimental groups for MAO activity detection. The zebrafish larvae were transferred to a 96-well plate with a pipette, and 16 wells were repeated in each group. The movement trajectory and distance of the zebrafish larvae were recorded using an automatic video tracking system (Videotrack). The trajectory recording parameters were set to light 5min + black 5min, the threshold was 0 (all movements were counted), the unit was mm, light 5min was used to stabilize the larvae, and the trajectory and movement distance of black 5min were analyzed. The experimental results are as follows (* 0.01＜P≤0.05, ** P≤0.01)):

1.5 Experimental Results

Zebrafish behavior is based on the distance of swimming to judge the degree of depression. Long swimming distance means low depression, short swimming distance means high depression. The model group can significantly inhibit the activity of zebrafish (P<0.05). According to the data of zebrafish activity trajectory in the figure, it can be seen that the activity trajectory and distance of zebrafish in the model group are significantly less than those in other groups. Compared with the model group, the positive drug group can significantly increase the activity of zebrafish (P<0.05). All three concentrations of the sample can significantly increase the activity of zebrafish (P<0.05). The behavioral data are basically consistent with the quantitative data of MAO.

2.1 Zebrafish behavioral results:

The zebrafish behavioral results are shown in Table 6:

Table 6 Results of activity distance of zebrafish larvae

From the results in Table 6, it can be seen that the activity distance of the reserpine model group was significantly reduced compared with the control group, which was only 74% of the control group, a decrease of 26%; the activity distance of the positive drug Toloxatone was significantly increased, which increased by 11.2% compared with the control group; and increased by 50% compared with the model group; while the activity distance of zebrafish in the three groups of limonene nicotine atomized liquid was significantly increased, and the activity distance of the low concentration group was significantly increased compared with the control group.

The activity distance of zebrafish in the medium concentration group increased significantly by 185%, which was 123% higher than that of the positive drug; the activity distance of the medium concentration group increased significantly by 141% compared with the control group, which was 127% higher than that of the positive drug; the activity distance of the high concentration group increased significantly by 145% compared with the control group, which was 196% higher than that of the positive drug. These results suggest that reserpine can cause a significant decrease in the activity distance of zebrafish, leading to the appearance of depressive symptoms, and the positive drug Toloxatone can significantly increase the activity distance of zebrafish, which can counteract the depressive symptoms of zebrafish. The activity distance of zebrafish in the three groups of limonene nicotine aerosol solution increased significantly, which was stronger than the effect of the positive drug Toloxatone, suggesting that in the zebrafish depression model caused by reserpine, the effect of limonene nicotine aerosol solution is stronger than that of the antidepressant drug Toloxatone.

The electronic cigarette atomizer liquid prepared by the technical solution of the present invention has the effects of inhibiting the activity of monoamine oxidase and resisting depression, has high safety, and can be used as an atomizer for resisting depression.

Industrial Applicability

The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

.An electronic cigarette atomizer liquid with antidepressant effect, characterized in that its preparation components include, by weight: 0.1 to 4 parts of limonene, 0.1 to 5 parts of nicotine, 1 to 8 parts of menthol, 70 to 80 parts of propylene glycol, and 10 to 30 parts of glycerol.
The electronic cigarette atomizer liquid with antidepressant effect according to claim 1 is characterized by comprising 0.5 parts of limonene, 4 parts of nicotine, 3 parts of menthol, 72.5 parts of propylene glycol, and 20 parts of glycerol.
The method for preparing the electronic cigarette atomizer liquid having an antidepressant effect as claimed in claim 1 or 2, characterized in that it comprises the following steps:

S1. Weigh each component according to the weight ratio of the formulated components;

S2. The components are sequentially added to the centrifuge tube, stirred and mixed at a stirring rate of 200 rad / min;

S3. Soaking at 40°C for 480 minutes;

S4. Heat to 60°C, keep for 20 minutes and then cool to room temperature;

S5. Filter out impurities and you are done.