# Hypnotic Effect ofA. absinthium Hydroalcoholic Extract in Pentobarbital-Treated Mice

Authors: Hassan Rakhshandeh; Amirhossein Heidari; Ali Mohammad Pourbagher-Shahri; Roghayeh Rashidi; Fatemeh Forouzanfar
Journal: Neurology Research International
 (2021)

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## Abstract

Background. Current drugs used in the management of insomnia are associated with side effects. The use of medicinal herbs for insomnia treatment has recently been suggested. Objective. The present study aimed to determine the hypnotic activity of the hydroalcoholic extract of Artemisia absinthium (A. absinthium) in mice. Method. The toxicity of A. absinthium extract is assessed by their lethal dose 50% (LD50), and cytotoxicity evaluation was also done with PC12 cell lines by MTT assay. A. absinthium extract (25, 50, 100, and 200 mg/kg) and 3 fractions (n-butanol fraction (NBF), ethyl acetate fraction (EAF), and aqueous fraction (AQF)) were administered intraperitoneally30 minutes before 30 mg/kg pentobarbital intraperitoneal injection; after that, the sleeping time and sleep latency were recorded. Results. The LD50 value was 2.4 g/kg. The extracts tested showed no negative effect on the proliferation of PC12 cells. A. absinthium extract increased the duration of pentobarbital-induced sleep at doses of 100 and 200 mg/kg (P<0.01-P<0.001). Similarly, AQF, EAF, and NBF at 200 mg/kg could increase sleep duration (P<0.05). The sleep latency was decreased by A. absinthium extract at doses of 100 and 200 mg/kg (P<0.05-P<0.01), AQF (P<0.05), and EAF (P < 0.05). Besides, flumazenil reversed the hypnotic effect of A. absinthium extract (P<0.05). Conclusion. A. absinthium extract probably demonstrated sleep-enhancing effects by regulating GABAergic system.


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## Body

## 1. Introduction

Insomnia has a negative impact on the quality of life leading to impairment in memory, mood disorders, and longer reaction [1–3]. Controlled-release melatonin, doxepin, z-drugs (zolpidem, eszopiclone, and zaleplon), and the orexin receptor antagonist suvorexant are major drug classes used to treat insomnia. Although these hypnotic drugs may improve sleep quality, they are associated with unwanted side effects [4]. The use of herbal products, as one element of complementary and alternative medicine, is increasing globally [5]. Artemisia absinthium (A. absinthium) (Wormwood) is an important perennial shrubby medicinal plant that is globally distributed in Europe, Asia, Middle East, and North Africa [6]. A. absinthium is named by numerous common names. It is named as wormwood, green ginger, absinthe, absinthium, in English; Genepi in Latin; Absinth, Wermut in German; Damseeh and Afsanteen in Arabic [6]. A. absinthium is employed in folk medicine as a febrifuge, antiseptic, antispasmodic, anthelmintic, cardiac stimulant, for repairing deteriorating mental function and memory improvement [7, 8]. Recently, it has shown various pharmacological effects including neuroprotective [9], antidiabetic [10], and hepatoprotective [11] properties. Other Artemisia species such as A. persica showed antianxiety [8] effects. A. absinthium contains compounds such as terpenoids (e.g., linalool), flavonoids (e.g., quercetrin) that these constituents have been reported to have hypnotic effects [12].The barbiturate pentobarbital binds to gamma-aminobutyric acid type A (GABA(A)) receptors to potentiate the effects of GABA [13]. Pentobarbital-induced hypnosis in mice is a popular animal model for screening of compounds with suspected sedation/hypnotic properties [14]. Therefore, the current study was intended to estimate the sleep-prolonging effect of A. absinthium and its fractions. Furthermore, the possible neurotoxicity of the plant was evaluated using a rat pheochromocytoma PC12 cell line.


## 2. Material and Methods

### 2.1. Reagents and Chemicals

Pentobarbital sodium, flumazenil, 3-(4,5-dimethyl thiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT), and penicillin-streptomycin were bought from Sigma (USA). Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), and other cell culture material were bought from Gibco Life Technologies (USA). Diazepam was obtained from Chemidarou Company (Iran). Tween 80 was purchased from Merck (Germany).


### 2.2. Plant and Extract

Aerial parts ofA. absinthium were collected from the Allah Akbar mountain (Khorasan Razavi Province of Iran). The plant was identified in the Herbarium of Khorasan Razavi Agricultural and Natural Resources Research Center. A voucher specimen (No. 11856) has been deposited in this institute. The powder of the aerial parts of A. absinthium was percolated with ethanol 70% for 72 hr. After extraction, the solvents were removed [15]. A part of dried A. absinthium was further subjected to solvent-solvent extraction for three fractions, namely, n-butanol fraction (NBF), ethyl acetate fraction (EAF), and aqueous fraction (AQF). The yield of AQF, NBF, and EAF was 55%, 18%, and 27%, respectively. A. absinthium fractions were dried on a water bath. The A. absinthium extract and its fractions were dissolved in normal saline comprising 1% Tween 80 [16].


### 2.3. Animals

Male albino mice weighing 25–35 g were maintained at 22 ± 1°C with 12 hr of light and 12 hr of dark and had free access to water and food. Experiments were carried out in accordance with the ethical guidelines approved by the Animal Care Use Committee of Mashhad University of Medical Sciences (981134).


### 2.4. Grouping and Treatment

A total of 66 adult male albino mice were divided into 6 groups as below:Group 1: Control (normal saline)Group 2: Positive control or Diazepam (3 mg/kg)Groups 3–6: Treatment group or different doses ofA. absinthium extract (25, 50, 100, and 200 mg/kg)Groups 7–9: Treatment group with AQF, EAF, and NBF (200 mg/kg)Group 10: Diazepam (3 mg/kg) plus flumazenil (2 mg/kg)Group 11:A. absinthium extract at a dose of 200 mg/kg plus flumazenil (2 mg/kg)


### 2.5. All Agents Were Administrated Intraperitoneally

Thirty min after the intraperitoneal administration of the agents, pentobarbital (30 mg/kg IP) was administrated to induce sleep. Time elapsed between the administrations of pentobarbital until loss of righting reflex and immobility was considered as the sleep latency, while the time from the loss to its recovery was considered the sleeping time. Mice were given flumazenil (2 mg/kg) 30 min before diazepam orA. absinthium extract administration [17].


### 2.6. Toxicity Assessment

The possible neurotoxicity ofA. absinthium extract and its fractions was tested on PC12 cells. Cells were seeded in 96-well plates in DMEM culture medium supplemented with 10% FBS and 1% penicillin streptomycin. After 24 hours, the culture medium was changed to fresh one having vehicle (1% Tween 80) or A. absinthium extract (200, 400, and 800 μg/mL) and its fractions (800 μg/mL). After 24 hours, the MTT (0.5 mg/mL in final volume) was added to culture medium of each well and incubated for 2 hours. Afterwards, the medium was discarded and DMSO was added to each well to dissolve any formazan crystals formed. The absorption of dye was measured at 570 nm [15]. For the determination of LD50, a series of doses of A. absinthium extract were administered intraperitoneally to mice. The number of deaths in each group within 24 hours was recorded. The maximum dose which did not cause death and the minimum dose causing death of one animal were noted. The average of these two doses was defined as the median lethal dose [18].


### 2.7. Data Analysis

All results are presented as mean ± SEM. Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Tamhane’s T2 post-hoc test. Values withp<0.05 were considered as statistically significant.


## 2.1. Reagents and Chemicals

Pentobarbital sodium, flumazenil, 3-(4,5-dimethyl thiazole-2yl)-2,5-diphenyl tetrazolium bromide (MTT), and penicillin-streptomycin were bought from Sigma (USA). Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), and other cell culture material were bought from Gibco Life Technologies (USA). Diazepam was obtained from Chemidarou Company (Iran). Tween 80 was purchased from Merck (Germany).


## 2.2. Plant and Extract

Aerial parts ofA. absinthium were collected from the Allah Akbar mountain (Khorasan Razavi Province of Iran). The plant was identified in the Herbarium of Khorasan Razavi Agricultural and Natural Resources Research Center. A voucher specimen (No. 11856) has been deposited in this institute. The powder of the aerial parts of A. absinthium was percolated with ethanol 70% for 72 hr. After extraction, the solvents were removed [15]. A part of dried A. absinthium was further subjected to solvent-solvent extraction for three fractions, namely, n-butanol fraction (NBF), ethyl acetate fraction (EAF), and aqueous fraction (AQF). The yield of AQF, NBF, and EAF was 55%, 18%, and 27%, respectively. A. absinthium fractions were dried on a water bath. The A. absinthium extract and its fractions were dissolved in normal saline comprising 1% Tween 80 [16].


## 2.3. Animals

Male albino mice weighing 25–35 g were maintained at 22 ± 1°C with 12 hr of light and 12 hr of dark and had free access to water and food. Experiments were carried out in accordance with the ethical guidelines approved by the Animal Care Use Committee of Mashhad University of Medical Sciences (981134).


## 2.4. Grouping and Treatment

A total of 66 adult male albino mice were divided into 6 groups as below:Group 1: Control (normal saline)Group 2: Positive control or Diazepam (3 mg/kg)Groups 3–6: Treatment group or different doses ofA. absinthium extract (25, 50, 100, and 200 mg/kg)Groups 7–9: Treatment group with AQF, EAF, and NBF (200 mg/kg)Group 10: Diazepam (3 mg/kg) plus flumazenil (2 mg/kg)Group 11:A. absinthium extract at a dose of 200 mg/kg plus flumazenil (2 mg/kg)


## 2.5. All Agents Were Administrated Intraperitoneally

Thirty min after the intraperitoneal administration of the agents, pentobarbital (30 mg/kg IP) was administrated to induce sleep. Time elapsed between the administrations of pentobarbital until loss of righting reflex and immobility was considered as the sleep latency, while the time from the loss to its recovery was considered the sleeping time. Mice were given flumazenil (2 mg/kg) 30 min before diazepam orA. absinthium extract administration [17].


## 2.6. Toxicity Assessment

The possible neurotoxicity ofA. absinthium extract and its fractions was tested on PC12 cells. Cells were seeded in 96-well plates in DMEM culture medium supplemented with 10% FBS and 1% penicillin streptomycin. After 24 hours, the culture medium was changed to fresh one having vehicle (1% Tween 80) or A. absinthium extract (200, 400, and 800 μg/mL) and its fractions (800 μg/mL). After 24 hours, the MTT (0.5 mg/mL in final volume) was added to culture medium of each well and incubated for 2 hours. Afterwards, the medium was discarded and DMSO was added to each well to dissolve any formazan crystals formed. The absorption of dye was measured at 570 nm [15]. For the determination of LD50, a series of doses of A. absinthium extract were administered intraperitoneally to mice. The number of deaths in each group within 24 hours was recorded. The maximum dose which did not cause death and the minimum dose causing death of one animal were noted. The average of these two doses was defined as the median lethal dose [18].


## 2.7. Data Analysis

All results are presented as mean ± SEM. Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Tamhane’s T2 post-hoc test. Values withp<0.05 were considered as statistically significant.


## 3. Results

### 3.1. Assessment of Toxicity

The highest dose ofA. absinthium extract that did not result in death in any mouse and the lowest dose that resulted in death in one mouse were 1.6 and 3.2 g/kg, respectively. The LD50 value of A. absinthium extract was 2.4 g/kg. Results showed no significant decrease in the proliferation of PC12 cells at the concentrations of 100, 200, 400, and 800 μg/ml of the extract and 800 μg/ml of the fractions (Table 1).Table 1
Effects ofA. absinthium on viability of PC-12 cell. Values are expressed as the mean ± SEM (n = 4).
SalineExtract 200Extract 400Extract 800EAF 800NBF 800AQF 800100.0 ± 2.19893.75 ± 1.31593.25 ± 1.79795.00 ± 1.08094.00 ± 1.47296.00 ± 0.707193.25 ± 1.377


### 3.2. Effects ofA. absinthium Extract on Pentobarbital-Induced Sleeping Behaviors in Mice

The results show that diazepam (3 mg/kg) andA. absinthium extract (100 and 200 mg/kg) significantly increased the sleep time compared with the control group (P<0.001, P<0.01, and P<0.001, resp.). Flumazenil (2 mg/kg) and A. absinthium extract (200 mg/kg) significantly reversed the effects of the diazepam (3 mg/kg) (P<0.001) (Table 2).Table 2
Effects ofA. absinthium extract (25, 50, 100, and 200 mg/kg) or diazepam (3 mg/kg) on duration of sleep. Values are expressed as the mean + SEM (n = 6). ∗∗p<0.01 and ∗∗∗p<0.001 vs. saline; p###<0.001 vs. the same group without flumazenil (2 mg/Kg).
SalineDiazepamDiazepam + flumazenilExtract 25Extract 50Extract 100Extract 200Extract 200+ flumazenil21.33 ± 1.90940.33 ± 1.022∗∗∗22.00 ± 0.6831###22.83 ± 0.945825.67 ± 1.68730.67 ± 2.275∗∗32.50 ± 1.176∗∗∗22.67 ± 1.453 ###As illustrated in Table3, diazepam (3 mg/kg) and A. absinthium extract (100 and 200 mg/kg) significantly reduced the sleep latency compared with the control group (P<0.001, P<0.05, and P<0.01, resp.). Flumazenil (2 mg/kg) and A. absinthium extract (200 mg/kg) significantly reversed the augmented effects of diazepam (3 mg/kg) in pentobarbital-treated mice (P<0.001 and P<0.05, respectively) (Table 3).Table 3
Effects ofA. absinthium extract (25, 50, 100, and 200 mg/kg) or diazepam (3 mg/kg) on the sleep latency. Values are expressed as the mean + SEM (n = 6). ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 vs. saline; p#<0.05, p###<0.001 vs. the same group without flumazenil (2 mg/kg).
SalineDiazepamDiazepam + flumazenilExtract 25Extract 50Extract 100Extract 200Extract 200 + flumazenil8.833 ± 0.47733.667 ± 0.2108∗∗∗8.333 ± 0.5578 ###8.667 ± 0.55787.833 ± 0.47735.833 ± 0.8333∗5.333 ± 0.7149∗∗8.5 ± 0.6#Effects ofA. absinthium extract fractions on pentobarbital-induced sleeping behaviors in mice are as follows:The results show that all fractions (200 mg/kg) significantly increased the sleep time compared with the control group (P<0.05) (Table 4). EAF and AQF (200 mg/kg) significantly reduced the sleep latency compared with the control group (P<0.05) (Table 5).Table 4
Effects of aqueous fraction (AQF), ethyl acetate fraction (EAF), or n-butanol fraction (NBF) ofA. absinthium extract on the duration of sleep. Values are expressed as the mean + SEM (n = 6). ∗p<0.05 vs. saline.
SalineEAFNBFAQF21.33 ± 1.90928 ± 1∗28.33 ± 1.16∗28 ± 1.4∗Table 5
Effects of aqueous fraction (AQF), ethyl acetate fraction (EAF), or n-butanol fraction (NBF) ofA. absinthium extract on sleep latency. Values are expressed as the mean + SEM (n = 6). ∗p<0.05 vs. saline.
SalineEAFNBFAQF8.833 ± 0.47735.8 ± 0.9∗6.5 ± 0.675.8 ± 0.7∗


## 3.1. Assessment of Toxicity

The highest dose ofA. absinthium extract that did not result in death in any mouse and the lowest dose that resulted in death in one mouse were 1.6 and 3.2 g/kg, respectively. The LD50 value of A. absinthium extract was 2.4 g/kg. Results showed no significant decrease in the proliferation of PC12 cells at the concentrations of 100, 200, 400, and 800 μg/ml of the extract and 800 μg/ml of the fractions (Table 1).Table 1
Effects ofA. absinthium on viability of PC-12 cell. Values are expressed as the mean ± SEM (n = 4).
SalineExtract 200Extract 400Extract 800EAF 800NBF 800AQF 800100.0 ± 2.19893.75 ± 1.31593.25 ± 1.79795.00 ± 1.08094.00 ± 1.47296.00 ± 0.707193.25 ± 1.377


## 3.2. Effects ofA. absinthium Extract on Pentobarbital-Induced Sleeping Behaviors in Mice

The results show that diazepam (3 mg/kg) andA. absinthium extract (100 and 200 mg/kg) significantly increased the sleep time compared with the control group (P<0.001, P<0.01, and P<0.001, resp.). Flumazenil (2 mg/kg) and A. absinthium extract (200 mg/kg) significantly reversed the effects of the diazepam (3 mg/kg) (P<0.001) (Table 2).Table 2
Effects ofA. absinthium extract (25, 50, 100, and 200 mg/kg) or diazepam (3 mg/kg) on duration of sleep. Values are expressed as the mean + SEM (n = 6). ∗∗p<0.01 and ∗∗∗p<0.001 vs. saline; p###<0.001 vs. the same group without flumazenil (2 mg/Kg).
SalineDiazepamDiazepam + flumazenilExtract 25Extract 50Extract 100Extract 200Extract 200+ flumazenil21.33 ± 1.90940.33 ± 1.022∗∗∗22.00 ± 0.6831###22.83 ± 0.945825.67 ± 1.68730.67 ± 2.275∗∗32.50 ± 1.176∗∗∗22.67 ± 1.453 ###As illustrated in Table3, diazepam (3 mg/kg) and A. absinthium extract (100 and 200 mg/kg) significantly reduced the sleep latency compared with the control group (P<0.001, P<0.05, and P<0.01, resp.). Flumazenil (2 mg/kg) and A. absinthium extract (200 mg/kg) significantly reversed the augmented effects of diazepam (3 mg/kg) in pentobarbital-treated mice (P<0.001 and P<0.05, respectively) (Table 3).Table 3
Effects ofA. absinthium extract (25, 50, 100, and 200 mg/kg) or diazepam (3 mg/kg) on the sleep latency. Values are expressed as the mean + SEM (n = 6). ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.001 vs. saline; p#<0.05, p###<0.001 vs. the same group without flumazenil (2 mg/kg).
SalineDiazepamDiazepam + flumazenilExtract 25Extract 50Extract 100Extract 200Extract 200 + flumazenil8.833 ± 0.47733.667 ± 0.2108∗∗∗8.333 ± 0.5578 ###8.667 ± 0.55787.833 ± 0.47735.833 ± 0.8333∗5.333 ± 0.7149∗∗8.5 ± 0.6#Effects ofA. absinthium extract fractions on pentobarbital-induced sleeping behaviors in mice are as follows:The results show that all fractions (200 mg/kg) significantly increased the sleep time compared with the control group (P<0.05) (Table 4). EAF and AQF (200 mg/kg) significantly reduced the sleep latency compared with the control group (P<0.05) (Table 5).Table 4
Effects of aqueous fraction (AQF), ethyl acetate fraction (EAF), or n-butanol fraction (NBF) ofA. absinthium extract on the duration of sleep. Values are expressed as the mean + SEM (n = 6). ∗p<0.05 vs. saline.
SalineEAFNBFAQF21.33 ± 1.90928 ± 1∗28.33 ± 1.16∗28 ± 1.4∗Table 5
Effects of aqueous fraction (AQF), ethyl acetate fraction (EAF), or n-butanol fraction (NBF) ofA. absinthium extract on sleep latency. Values are expressed as the mean + SEM (n = 6). ∗p<0.05 vs. saline.
SalineEAFNBFAQF8.833 ± 0.47735.8 ± 0.9∗6.5 ± 0.675.8 ± 0.7∗


## 4. Discussion

Based on experimental result,A. absinthium extract reduced the sleep latency and augmented the total sleep in pentobarbital-induced sleeping mice. Previous studies have shown that a wide range of herbal medicine ingredients including alkaloids, flavonoids (e.g., quercetrin and luteolin), terpenoids (e.g., linalool), and steroids have hypnotic properties [12]. A. absinthium contains numerous phytochemical ingredients, namely, lactones, terpenoids (e.g., γ-terpinene, 1,4-terpeniol, trans-thujone, cadinene camphene, bornyl acetate, trans-sabinyl acetate, myrcene, guaiazulene, chamazulene, camphor, and linalool), essential oils, tannins, organic acids, resins, and phenols. The plant also have flavonoids (e.g., quercetin), flavonoid glycosides such as quercitin-3-O-D-glucoside, isorhamnetin-3-O-rhamnose glucoside, quercetin-3-O-rhamnoglucoside, isoquercitrin, and isorhamnetin-3-O-glucoside, and phenolic acids (coumaric, chlorogenic, syringic, salicylic, and vanillic acids) [6, 19, 20]. Benzodiazepines such as diazepam enhance the effect of the neurotransmitter gamma-aminobutyric acid (GABA) at the GABAA receptor and thereby increase pentobarbital-induced sleeping time [21]. Indeed, GABA opens the Cl− channels accompanied by a membrane hyperpolarization that cause CNS depression and sedative and hypnotic action [22, 23]. Therefore, drugs that affect these systems can be considered in the treatment of insomnia. Accordingly, we observed that pretreatment of mice with flumazenil (as the antagonist of GABAA benzodiazepine receptor) reduces the prolonged sleep effect of diazepam. Furthermore, we found that flumazenil reverses the hypnotic effect of A. absinthium extract. We prepared three fractions from A. absinthium extract: the AQF, which contains solubilizing the polar agents and water-soluble plant ingredients (e.g., glycosides, tannins, and quaternary alkaloids); EAF, which contains ingredients with intermediate polarity; and the NBF, which contains nonpolar agents like sterols, alkanes, and some terpenoids [14]. In our study, all fractions augmented sleep duration, suggesting that the extract and its various fractions may have bioactive constituents with hypnotic property. To evaluate the fact that the hypnotic effect is associated with no negative impact on neurons, we utilized PC12 cells. No cytotoxic effect for A. absinthium extract was found at 24 h period in PC12 cells. Acute toxic study demonstrated the lethal dose (LD (50)) of A. absinthium extract was 2.4 g/kg. Based on these results, A. absinthium possesses hypnotic activities in a relatively safe dose range.


## 5. Conclusion

Taken together, all the data presented here demonstrate thatA. absinthium extract increased hypnotic effects in pentobarbital-treated mice. The GABAergic system might be involved in the mechanisms of these effects. Further studies are needed to support this evidence.



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*Source: 5521019-2021-04-22.xml*