Effects of Coenzyme Q10 Sleep Aid Emulsion on the Reflex of Turning over in Mice

Abstract :OBJECTIVE: To study the effects of Coenzyme Q10 sleep aid emulsion on the reflex of turning over in mice. Methods: 99 SPF-grade Kunming female mice were randomly divided into 9 groups: saline group, high-dose group, medium-dose group, low-dose group, melatonin-deficient group, 5-hydroxytryptophan-deficient group, glutamate-deficient group, coenzyme Q10-deficient group and osmosis-deficient group, 10 mice in each group. After the mice in each group were dehairing and kept without food or water for 8 h, the samples were smeared on the back of their hairless places, and after 50 min of drug administration, the samples were injected with 45 mg/(kg-BW) of sodium pentobarbital solution intraperitoneally for the threshold of sodium pentobarbital. After 50 min of drug administration, mice were injected intraperitoneally with 45 mg/(kg-BW) pentobarbital sodium solution for suprathreshold pentobarbital sodium sleep test.

 

RESULTS: The high and medium dose groups of coenzyme Q10 sleep aid emulsion could prolong the sleep time of mice, and there was a significant difference compared with that of the blank group (P < 0.05), indicating that the coenzyme Q10 sleep aid emulsion had a certain sleep-aiding effect; and the medium dose group could prolong the sleep time of mice and shorten the latency period of sleep in the absence of ghrelin and transdermal absorption enhancers (P < 0.01). Conclusion Conclusion: Coenzyme Q10 sleep-enhancing emulsion has the effect of promoting sleep in mice.

 

In the rapid development of society, the accelerated pace of life, which brings insomnia, anxiety and other problems; in daily life, sleep accounts for one-third of the time, when sleep problems, easily caused by the work, life, physical and mental health and other aspects of the impact, and in serious cases, and even life-threatening [1]. Coenzyme Q10 sleep aid emulsion is composed of Coenzyme Q10, glutamine, melatonin, 5-hydroxytryptophan, tyrosine and other ingredients.

 

Among them, Coenzyme Q10, also known as ubiquinone (molecular weight 863.36), is mainly used in the prevention and treatment of cardiovascular diseases. Experiments at home and abroad have proved that Coenzyme Q10 can improve the sleep disorders caused by Parkinson's disease after 3 months of treatment with a normal dose of Coenzyme Q10 (60 mg per day), and that it can improve the cognitive function, anxiety, and sleep disorders of the patients after 6 months of treatment. It can prevent the degeneration of dopaminergic neurons caused by MPP+ and protect dopaminergic neurons. Dopamine, as an important neurotransmitter in the brain, is involved in the active neuromodulation of sleep, which suggests that coenzyme Q10 may have a certain effect on sleep disorders. The results show that Coenzyme Q10 may have some effects on sleep disorders [2].

 

Melatonin is a neuroendocrine hormone synthesized and secreted by the pineal gland, which can achieve the effect of sleep aid through the specific melatonin receptor-mediated action. However, melatonin has a short half-life, poor absorption, and is prone to cause a first-off effect when taken orally [3], and the skin, as the largest organ in the human body, has a transcutaneous delivery system that can significantly reduce the elimination of melatonin and improve its bioavailability. Tyrosine can promote the absorption of melatonin by the skin and the synthesis of melatonin in the body (CN 105193794B); tyrosine can effectively relieve mood, improve drug-resistant depression, agitation, allergy, anxiety, etc., and relax the mind.

 

5-Hydroxytryptophan is an important intermediate in the metabolism of tryptophan to 5-hydroxytryptamine, which is a neurological effector, and its level has a certain degree of influence on human health, including sleep problems. In human and animal experiments, it was found that patients who took 5-hydroxytryptophan could improve the problem of mild insomnia [4]. Glutamine has a certain regulatory effect on patients with neurasthenia; at the same time, it can stabilize mood, reduce anxiety and tension, and improve sleep [5]. As a natural physiological barrier [6], the skin can prevent drugs from entering the body, so most of the drugs have a low transdermal permeability, so transdermal absorption enhancers are needed to help the drugs enter the skin and then enter the body to achieve drug efficacy. In this study, we investigated the effects of Coenzyme Q10 sleep aid emulsion on the reflex of mice, and provided the parameters for the follow-up experimental study.

 

1 Materials and Methods

1.1 Experimental materials

1.1.1 Experimental animals: Kunming breed mice, female, weighing 18~22 g, 6~8 weeks, SPF grade, provided by the Laboratory Animal Center of Southern Medical University, with free access to water and food during the feeding period. License No.: SCXK(GD)2016-0041.

 

1.1.2 Drugs.

Sleeping Emulsion was supplied by Coenzyme Q10 Joint Research Center of Guangdong Medical University-Guangdong Runhe Biotechnology Co., Ltd. and the raw materials include Coenzyme Q10, Melatonin, Tyrosine, 5-Hydroxytryptophan, Glutamine, Glycerin, Anhydrous Ethanol (Tianjin Damao Chemical Reagent Factory, Analytically Pure), Menthol, Nitrogen Ketone, Lavandula Angustifolia Essential Oil, Pearl Extract, Dipotassium Glycyrrhizinate, Vitamin B6, Phenoxyethanol, Almond oil, stearic acid, Ganoderma lucidum spore oil.

 

1.1.3 Reagents and main instruments.

Pentobarbital Sodium (Merck), physiological saline (Cinnabar Pharmaceutical Co., Ltd.); electronic balance (Shenzhen Mobil Electronics Co., Ltd.); hair removal cream (Vitamin Co., Ltd.); shaving machine FC5809 (Shanghai Feike Electrical Appliances Co., Ltd.); constant-temperature heating magnetic stirrer (BONSIE Instrument Technology (Shanghai) Co. ), Timer.

1.2 Experimental methods

1.2.1 Subgroups: see Table 1.

1.2.2 Preparation of the formulation.

Dissolve melatonin in 5 mL of glycerol and mix with 1 mL of anhydrous ethanol, 0.25 g of menthol, 1.5 g of azathioprine, and 0.25 g of lavender essential oil.

Dissolve 0.15 g of dipotassium glycyrrhizinate and 0.15 g of vitamin B6 in 10 mL of pearl aqueous extract.

Dissolve tyrosine and 0.15 g phenoxyethanol in 15 mL of distilled water and mix with the solution to be used (1) and (2).

④ Use 5 mL of almond oil, add 2 g of stearic acid 21 and 2 g of stearic acid, heat to dissolve, set aside.

⑤ Dissolve 0.25 g of Coenzyme Q10, glutamine and 5-hydroxytryptophan in 2.5 mL of Ganoderma lucidum spore oil and mix well with ④. ⑥ Emulsify ③ and ⑤ and mix well and store in separate packages, labeled with date, number and name.

 

1.2.3 Laboratory animal handling.

Ninety-nine mice were shaved on the back, kept without food or water for 8 h, weighed and randomly divided into 9 groups of 10 mice each, and the excess 9 mice were kicked out.

 

1.2.4 Effects on the sodium pentobarbital suprathreshold sleep test/

Fifteen drops of the sample were slowly applied to the hairless area on the back of mice and absorbed by massaging within 3 min, and the time of administration (T.D.) was recorded. The time of injection (T.R) was recorded at the time of intraperitoneal injection of sodium pentobarbital (0.1 mL/10 g of body weight) at a dose of 45 mg/(kg-BW) 50 min after the application of the drug to the mice, the time of disappearance of the reversal reflex was recorded at the time of disappearance of the reversal reflex (T.S) within 15 min, and the time of awakening from sleep was recorded when the reversal reflex was restored (T.W). From the above indexes, the sleep latency (T.S-T.R, min) and sleep duration (T.W-T.S, min) were calculated.

Table 1 Experimental groups and formulations

 

groups	Sleeping Aid Emulsion - Formulation
G1	saline (medicine)
G2	High dose group (melatonin, tyrosine, glutamine, 5-hydroxytryptophan: 6%, 6%, 4%, 2%)
G3	Medium dose group ( melatonin, tyrosine, glutamine, 5-hydroxytryptophan :3%, 3%, 2%, 1% )
G4	Low-dose group (melatonin, tyrosine, ghrelin, 5-hydroxytryptophan :1.5% , 1.5%, 1%, 0.5% )
G5	Melatonin-deficient group (tyrosine, glutamine, 5-hydroxytryptophan: 3%, 2%, 1%)
G6	5-Hydroxytryptophan Deficiency Group (melatonin, tyrosine, glutamine: 3%, 3%, 2%)
G7	Glutathione deficiency group (melatonin, tyrosine, 5-hydroxytryptophan: 3%, 3%, 1%)
G8	Group lacking excipients (Dipotassium glycyrrhizinate, Vitamin B6  , Pearl Aqua, Coenzyme Q10  , Ganoderma lucidum spore oil).
G9	Deficiency of osmotic stimulant group (deficiency of menthol, azathioprine)

 

1.2.5 Criteria for determining sleep.

Sleep was indicated by the disappearance of the reflex of turning over, when the mice were in dorsal recumbency and could not turn over for more than 60 seconds, it was considered that the reflex of turning over had disappeared and they entered into sleep. The sleep latency period was the time from the intraperitoneal injection of sodium pentobarbital to the disappearance of the reflex, and the sleep duration period was the time from the disappearance of the reflex to the awakening.

 

1.3 Statistical methods    

One-way analysis of variance (ANOVA) was performed using SPSS 23.0 software, and all measurements were expressed as mean ± standard deviation (x- ± s), and the t-test was used to determine the statistical significance of the differences between the groups, with P < 0.05 as statistically significant; graphs were prepared using Graphpad prism 6.

2 Results

2.1 Effect of sleep aid emulsions on sleep latency of suprathreshold doses of sodium pentobarbital (Table 2, Figure 1)  

In the sodium pentobarbital sleep latency experiment, after the injection of sodium pentobarbital solution, the neck of the mice appeared to be tilted back, turning reflex gradually disappeared, and the mice entered the sleep state, and there was no significant difference between the groups. However, the sleep latency time of the G5-G9 group was shortened for a certain period of time compared with that of the G1 group (blank) and the G3 group. This indicates that the addition of melatonin, 5-hydroxytryptophan, glutamine, transdermal absorption enhancer and other ingredients in the sleep aid emulsion will prolong the sleep latency time, of which dipotassium glycyrrhizinate, Ganoderma lucidum spore oil and other excipients have the smallest effect.

 

Table 2 Effect of sleep aid emulsions on sleep duration of suprathreshold doses of sodium pentobarbital (x- ± s,n = 10)

serial number	Number of sleeping animals (n)	sleep latency (min)	Sleep duration (min)
G1	7	7.3 ± 6.651	16.5 ± 14.714
G2	10	9.8 ± 12.291	40.7 ± 17.263 ∗ ∗
G3	10	7.7 ± 3.302	34.4 ± 7.648 ∗
G4	9	9.7 ± 10.467	29.4 ± 21.093
G5	7	3.9 ± 2.923	17.1 ± 13.153
G6	9	4.2 ± 2.251	25.2 ± 11.094
G7	10	4.5 ± 1.269	40.3 ± 25.743 ∗ ∗
G8	10	6 ± 4.989	28 ± 12.266
G9	9	4.1 ± 1.595	42.9 ± 27.795 ∗ ∗

 

Note:Compared with the blank control group , ∗ P <0.05 , ∗∗ P <0.01

 

2.2 Effect of sleep aid emulsions on sleep duration of suprathreshold doses of sodium pentobarbital (see Table 2, Figure 2)  

In the experiment of sleep duration of suprathreshold dose of sodium pentobarbital, compared with the G1 group, the G2, G7 and G9 groups had highly significant differences (P < 0.01), and the other groups prolonged the sleep duration of the mice, but there was no significant difference (P > 0.05), which indicated that the sleep-aiding emulsions had a certain sleep-aiding effect, and prolonged the sleep duration of the mice. Compared with the G3 group, when glutamine, transdermal absorption enhancer and 5-hydroxytryptophan were not added to the emulsion, the sleep duration of mice was prolonged, and 5-hydroxytryptophan had almost no effect on it; when melatonin, pearl aqueous extract and Ganoderma lucidum spore oil were not added to the emulsion, the sleep duration of mice was lowered, and melatonin had a greater effect on it. It was shown that the addition of transdermal absorption enhancer and glutamine in the sleep aid emulsion decreased the sleep duration of mice, while the absence of melatonin decreased the sleep duration of mice.

 

3 Conclusion and discussion

According to the relevant research, about 27% of the world's people suffer from insomnia, long-term insomnia may lead to physical and mental health, affecting the life and work, the current effective treatment is medication, but the side effects are large [7]. In this experiment, we observed whether Coenzyme Q10 sleep aid could prolong the sleep time of sodium pentobarbital through the suprathreshold dose of sodium pentobarbital sleep test. The study shows that Coenzyme Q10 sleep aid emulsion can prolong the sleep time of mice in the high dose and medium dose groups, compared with the blank group, there is a significant difference (P < 0.05), indicating that Coenzyme Q10 sleep aid emulsion has a certain effect on sleep.

 

When glutamine, 5-hydroxytryptophan, melatonin, and tyrosine were used at medium doses in the G5-G9 group, it was found that the absence of glutamine and transdermal absorption enhancers significantly prolonged the sleep time (P < 0.01) and shortened the sleep latency of the mice, which indicated that this ingredient could not synergize with the other ingredients and might even be antagonistic to them.

 

In the absence of melatonin, the duration of sleep was similar to that of the blank group, and it was inferred that melatonin (MT) was the main component of sleep aid. Generally, small lipophilic molecules with relative molecular mass less than 500 have high transdermal permeability, while MT has a small molecular weight (232.27) and can easily pass through the cuticle, and there are hydrophilic and lipophilic groups in its structure that make it easy to pass through the biofilm of the skin [8]; Glutathione has a larger molecular weight (602.89) and is difficult to pass through the skin barrier; and the use of azelone is the most effective in most studies, with 5% being the most effective. In most of the studies, 5% of azetone was the most effective [9,10].

 

In this experiment, 1.5% azidone was used, and its effect of promoting osmosis was not obvious, and it could not help the macromolecule of glutamine to enter the skin barrier, thus failing to achieve the sleep-aiding effect. In conclusion, Coenzyme Q10 sleep aid emulsion can prolong the sleep time of mice, in which melatonin is the main sleep aid ingredient, while glutamine in the emulsion can not achieve the sleep aid effect, and the combination with other ingredients can not be synergistic, or even antagonistic. The low amount of the osmotically promoted ingredients (nitrone and menthol) could not help the molecular weight ingredients to enter into the mice, which might be one of the factors affecting the effect of the sleep aid emulsions.

 

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