Evaluation of Sedative and Hypnotic Activity of Ethanolic Extract of Plumeria obtusa Flowers in Experimental Mice

Sleep disruptions are prevalent in older persons and frequently happen without any medical issues, they are increasingly being connected to negative health effects ^[1]. Nearly 50% of older individuals suffer from major conditions including sleep apnea and insomnia and many of them have trouble falling asleep and staying asleep as their sleep-wake cycle becomes disturbed with age ^[2]. These problems are addressed by sedative-hypnotic medications which relax the nervous system and make it easier to fall asleep and stay asleep. While hypnotics mimics natural sleep, sedatives lessen agitation without causing sleep. Sedative- hypnotics can provide these effects as central nervous system depressants without affecting mood or pain threshold^[3]. In India, insomnia is linked to serious comorbidities including diabetes and cardiovascular disease making it life life-threatening health concern ^[4]. More than 90% of Indians, according to recent research, do not get enough sleep, and many of them claim that their ability to function at work is adversely affected by their sleep, yet few of them seek medical assistance. Insomnia and other sleep disorders go untreated because of the social stigma associated with mental health issues. Traditional sedative-hypnotic drugs, such as benzodiazepines and non-benzodiazepine sedatives have been used extensively to treat insomnia; however, their efficacy is constrained by tolerance problems, addiction risks, and side effects, which has sparked interest in alternative therapy ^[3]. Herbal remedies are becoming more popular due to their low risk of adverse effects and possible ability to treat sleeplessness. Several plants including Plumeria obtusa, Valeriana officinalis, and Piper methysticum have demonstrated sedative-hypnotics properties. Rich in flavonoids such as apigenin, quercetin, and rutin Plumeria obtusa has long been used to treat insomnia and other conditions involving the central nervous system. It primarily acts on GABAergic pathways that are connected to calming effects. Its sedative-hypnotic properties are not well supported by scientific research, despite its well-established use in traditional medicine. Therefore, the purpose of this research is to investigate the efficacy of Plumeria obtusa flower extracts in sedative-hypnotic activity via experimental models ^[5].

METHODOLOGY:

Materials:

Chemicals such as Diazepam, and Pentobarbital of pure analytical grade were procured from E. Merck (India) Ltd, Mumbai, and all other chemicals and reagents of pure analytical grade were procured from local suppliers. Diazepam was used as a standard drug for comparison in behavioral experiments. The experiment was performed as described in the standard bibliography, kinds of literature, and textbooks.

Plant Collection and Extraction:

The flower of Plumeria obtusa used for the present studies was collected from Mangalore, Karnataka, in May 2024. It was authenticated by Dr. Siddaraju M.N. (Assistant Professor and Research Guide, Department of Botany, and University College Mangalore). Fresh Plumeria obtusa flowers were collected locally. The flowers were cut into small pieces. About 500g of the flower’s samples was macerated with 1.5 L of ethanol followed by frequent shaking for at least 3-7 days at room temperature. The obtained mixture was strained and filtered. The remaining residues were then discarded. The extracts obtained were stored in the refrigerator till further use ^[6].

Phytochemical Screening:

The ethanolic extract of Plumeria obtusa (POEE) was qualitatively investigated for the presence of different phytochemicals according to the standard procedures ^[7].

Dose Selection:

Dose was selected from a previous research work which is 100mg/kg and 200mg/kg respectively ^[8].

Experimental Animals:

The Swiss Albino mice (20-25 g) of either sex was used for this study. The animals were housed in colony cages and maintained under the standard environmental condition’s temperature    25± 2ºC, 12h light: 12 h dark cycle, and 50± 5 % relative humidity. They were fed with food and water ad libitum. The Institutional Animal Ethics Committee reviewed and approved the experimental protocol (Approval no: SCP/IAEC/F150/P217/2023). All the procedures were performed in accordance with Institutional Animal Ethics Committee considered as per the direction of the Committee for Control and Supervision of Experiments on Animals (CCSEA).

Experimental Design:

Hole Board Test:

The four groups of six mice each were randomly assigned to the following conditions Group I (Normal control: Vehicle), Group II (Diazepam: 3mg/kg, p.o.), Group III (Plumeria obtusa: 100 mg/kg, p.o.), Group IV (Plumeria obtusa: 200 mg/kg, p.o.). The mice were of both sexes and weighed (20-25g). Except for the vehicle-treated control group, all treatments were given orally for ten days. The animals were given medications 30 minutes before the sedative activity was evaluated on day 11^th.  A flat platform (60 x 30 cm) with 16 uniformly spaced holes was used to measure sedative activity. Each animal was given five minutes to investigate the platform, and the number of head dips into the holes was noted. During this time, the head dip counts were used to assess the sedative effects of treatments ^[9].

Potentiation of Pentobarbital-induced sleeping time test:

The mice of either sex weighing 20-25g were randomly assigned to four groups of six animals each including Group I (Normal control: Vehicle + Pentobarbital 30 mg/kg i.p.), Group II (Diazepam 3 mg/kg p.o. + Pentobarbital 30 mg/kg i.p.), Group III (Plumeria obtusa extract: 100 mg/kg p.o. + Pentobarbital 30 mg/kg i.p.), Group IV (Plumeria obtusa extract: 200 mg/kg p.o. + Pentobarbital 30 mg/kg i.p.). Except for the vehicle group, all treatments were given orally for ten days. Each animal was treated for 30 minutes before inducing hypnosis with pentobarbital on day 11^th. Pentobarbital was injected intraperitoneally to induce hypnosis and the animals were placed in a supine position on a pad that had been warmed to 37°C for 30 minutes after the injection. The time it took for the animals to lose the righting reflex was measured. This was verified by the fact that the animals could right themselves in less than a minute if they were placed on their backs. The hypnotic effect was assessed via loss of righting reflex ^[10].

Statistical Analysis:

The data were expressed as mean ±SEM and were analyzed by the one-way ANOVA (Analysis of variance) followed by Dunnett’s multiple-comparison test.

RESULTS:

The preliminary phytochemical screening of ethanolic extract of Plumeria obtusa (POEE) flowers showed the presence of flavonoids, tannins, alkaloids, phenolics, and glycosides. Because of these substances’ antioxidant qualities, result in possible pharmaceutical uses.

Hole Board Test: The Hole Board Test assesses sedative activity by measuring reduced exploratory behavior, such as decreased head-dipping, indicating CNS depression. It can also reflect anxiolytic effects if exploratory behavior increases. The diazepam showed a significant decrease in head-dip response. Treatment with Plumeria obtusa ethanolic extract (POEE) showed a dose-dependent reduction in head-dip response in comparison to control animals, suggesting that the extract possesses sedative activity rather than anxiolytic potentials as shown in Table No.1 and Fig No. 1,2 respectively.

       
           
       
Fig.No.1: Effect of POEE on Number of Head Dips in mice

The pentobarbital-induced sleeping time test evaluates hypnotic activity by examining compound effects on pentobarbital-induced sleep duration. Mice treated with the POEE showed a marked reduction in the onset of sleep and a dose-dependent prolongation of sleep duration compared to control animals, indicating enhanced sedative effects. The extract potentiated pentobarbital's hypnotic action, when compared to control group animals as shown in Table No. 2 and Fig No. 3,4respectively.

Table. No.2: Effect of POEE on the potentiation of pentobarbital-induced sleep time test in mice.

       
           
       
Fig.No.3: Effect of POEE on onset of sleep-in mice