Thiopental Sodium and diazepam were purchased from Incepta Pharmaceuticals Ltd (Dhaka, Bangladesh and Opsonin Pharma Ltd (Dhaka, Bangladesh). Distilled water and different reagents were obtained from BDH Chemicals Ltd (Dhaka, Bangladesh).

The seeds were obtained from 45–50 completely ripened fruits of the 4–5 Citrus limon plants, which were mature and aged 2–2.5 years, grown in fields close to Jahangirnagar University in Dhaka, Bangladesh, on 23rd October 2020. These were authenticated from Bangladesh National Herbarium, Mirpur, Dhaka.

The seeds collected from Citrus limon were carefully washed with distilled water to remove undesired natural parts. The seeds of the plants were dried under sunlight (temperature approximately 30–35 ${}^{\circ }$C) for two days and stored for seven days in an air-tight container. The dried seeds were ground to a coarse powder. The prepared fine powder was packed in a container and stored in cool, dark, and dry conditions to prepare fresh extracts before the investigation. A quantity of 500 gm of seed powder of Citrus limon was dissolved in 1000 mL of water in a glass compartment for 2–3 days, with standard shaking and mixing. Afterward, the entire mixture was passed through a fine, white cotton cloth. Further filtration was done using Whatman filter paper. Extracts were obtained by the evaporation of solvent that was kept in open space. The yield value of the extracts from the lemon seeds was 2.65% w/w [31].

Ninety Swiss albino mice (22–25 g) aged 6–7 weeks were obtained from Jahangirnagar University, Dhaka, Bangladesh. The animals were caged under standard natural conditions (22–25 ${}^{\circ }$C, humidity 60–70%, 12-hour light: 12-hour dull cycle). These were given a standard pellet diet, which was obtained from Jahangirnagar University, Dhaka. All protocols considered in our study, including animals, were approved by the Faculty of Allied Health Sciences Research Ethics Committee, Daffodil International University, Dhaka-1207, Bangladesh. (Ref: FAHSREC/DIU/2020/1006).

Qualitative assessment was done using colorimetric procedures with standard substances, indicating specific phytochemical groups, for example, glycosides, flavonoids, tannins, alkaloids, gums, steroids, saponins [32]. For carbohydrates, Molisch and Fehling’s tests were used. Identification of flavonoids was based on the standard flavonoid test. The Dragendroff’s, Mayer’s, and Hager’s tests were used for alkaloids. Potassium dichromate test, ferric chloride, and lead acetic acid derivation tests were pursued for tannins. For glycosides, Keller-Kiliani tests were performed. A frothing test was done for the presence of saponins. To detect steroids, the sulphuric acid analysis was performed. For detecting the presence of gum in the extracts, the Molisch test was further used.

Harborne’s (1973) method was used to analyze alkaloids: 5 g of the sample was weighed into a 250 mL beaker, and 200 mL of 10% acetic acid in ethanol was added, capped, and set aside for 4 hours. This was filtered, and the extract was concentrated to one-quarter of its original volume using a water bath. Dropwise additions of concentrated ammonium hydroxide to the extract were made until the precipitation was accomplished. The entire solution was allowed to settle and rinsed with weak ammonium hydroxide before being filtered. The residual alkaloid content was desiccated and weighed. Van-Burden and Robinson’s (1981) method was used to determine tannin: A 50 mL plastic bottle filled with 500 mg of the sample. In a mechanical shaker, 50 mL of distilled water was added and agitated for 1 hour. This was then filtered into a 50 mL volumetric flask and brought up to the required concentration. The filtrate was then pipetted into a test tube with 2 mL of 0.1 M FeCl${}_3$ in 0.1 N HCl and 0.008 M potassium ferrocyanide. Within 10 minutes, the absorbance was taken at 120 nm. Bohm and Kocipai Abyazan’s (1994) procedure was used for flavonoid determination: At room temperature, 10 g of the plant sample was extracted many times with 100 mL of 80 % aqueous methanol. Whatman filter paper No 42 was used to filter the entire solution (125 mm). The filtrate was then placed in a beaker and dried over a water bath before being weighed at a consistent weight [33, 34, 35].

The technique defined by Raihan MO et al. [36] was used to study the impact of the lemon seed extracts on sleeping time experiments induced by thiopental sodium. For such an objective, mice were grouped into six groups, with five (n = 5) mice in each group. Group I was used as a control and given distilled water (10 mL/kg b.w.), group II, as usual, diazepam (0.50 mg/kg b.w.) was used as standard and groups III, IV, V and VI were used for the seed extracts. Group II, III, IV, V and VI were administered orally using cannula, the standard drug diazepam (0.50 mg/kg b.w. and seed extracts in the doses: 25, 50, 100 and 200 mg/kg b.w. After thirty minutes, thiopental sodium (20 mg/kg b.w.) was injected intraperitoneally to all assortments to induce sleep. For monitoring adverse effects (if any), single mice from each group were placed on the table and examined. The length of sleep induced by thiopental sodium in animals was determined as indicated by the loss of their correct reflex. The percentages of effects were calculated using the following equation:

$$\text{Effect}(\%)=\frac{\text{The average duration of loss of righting reflex in the test group}}{\text{The average duration of loss of righting reflex in the control group}}\times 100$$

This investigation was carried out as described in the past by Uddin et al. [37]. A cage was utilized that has a size of 35 $\times$ 25 $\times$ 20 cm. In the enclosure, a segment was marked. At the height of 7.5 cm at the enclosure’s central focus, a hole with a diameter of 3 cm was created. Mice were administered with control, standard, or extract treatment and were put on one side of the platform. The number of passages taken by the mouse in each group through the hole from one chamber to another was then counted for 3 min at intervals 0, 30, 60, 90, and 120 min after administering the control, standard, and test sample (p.o.). This experiment was conducted within two days. Group I was set as control and given distilled water (10 ml/kg b.w.), and group II had taken diazepam (1 mg/kg, b.w.), which was used as standard. Groups III and IV were given seed extracts of 50 and 100 mg/kg body weight, respectively.

$$\text{Inhibition}(\%)=\frac{\text{Mean No. of movements (control)}-\text{Mean No. of movements (test)}}{\text{Mean No. of movements (control)}}\times 100$$

The test was performed as described by Kamei J et al. [38] with slight deviations. For the present test, a level platform of 90 cm $\times$ 90 cm in radius with 16 equitably separated holes was utilized. This stage likewise had a frame of 5 cm in height. Mice were grouped into four groups; control, standard, and test. Five (n = 5) mice were in each group. Group 1 (10 mL/kg b.w.) was listed for control and took distilled water. As standard, diazepam was provided to group II (1 mg/kg, b.w., p.o.). Groups III and IV independently took seed extracts in 50 and 100 mg/kg b.w. doses. The number of head dips in single mice into the holes was monitored for 10 min after the application.

$$\text{Inhibition}(\%)=\frac{\text{Mean No. of head dips (control) - Mean No. of head dips (test)}}{\text{Mean No. of head dips (control)}}\times 100$$

The method described by Gould TD et al. [39] was used for this analysis. The test device is made up of a plane 0.5 m${}^2$ field with a square progression. The squares on the other side are painted black and white. The experimental board is identical to a chessboard. Likewise, the mechanical system had a compartment height of 0.1 m. Mice were aligned into four groups. There were five (n = 5) mice in every group. Group I was assessed as control and given distilled water (10 mL/kg b.w.). Diazepam (1 mg/kg, b.w., p.o.), which was deemed standard, was given to Group II. Seed extract doses of 50 and 100 mg/kg b.w. were distinctly provided to Groups III and IV. The number of squares moved at any pace by the animals in each treated group was measured for 3 min at the time intervals 0, 30, 60, 90, and 120 min after the oral administration of test medication,

$$\text{Inhibition}(\%)=\frac{\text{Mean No. of movements}(\text{control})-\text{Mean No. of movements}(\text{test})}{\text{Mean No. of movements}(\text{control})}\times 100$$

Results are shown as mean $\pm$ SEM. Analyzes were carried out using one-way ANOVAs followed by Dunnett’s post hoc tests for sleeping time and hole board data. Two-way ANOVAs followed by Bonferroni’s post hoc tests were used for hole cross and open-field data. As a statistical result, p 0.05 was reported as significant.

A comprehensive range of chemical tests for identifying major classes of therapeutically significant compounds exhibited alkaloids, flavonoids, tannins, steroids, and cardiac glycosides in the aqueous extract of the Citrus limon seed. In contrast, saponins were found to be absent. Phytochemical compounds of Citrus limon aqueous extract of seeds are shown in Table 1. Quantitative estimation for certain phyto-constituents such as alkaloids, flavonoids and tannins in Citrus limon seed extract is also summarized in Table 2. The estimations found tannins to be present in good proportion compared to other molecules.

In the thiopental-induced hypnosis test, the plant extracts have shown a significant reduction in sleep onset and enhanced sleep duration in a dose-dependent manner. In this study, seed extract at doses of 25, 50, 100, and 200 mg/kg demonstrated a substantial decrease in sleep onset and increased sleeping time dependent on the dose (Table 3/p 0.05). The length of thiopental sodium-induced sleep time and its latency in experimental animals was effectively modulated by varying doses of the extract relative to controls (Figs. 1 and 2). As shown, the seed extract at 200 mg/kg had a maximal effect of 704% for lacking resolving reflex, while diazepam 0.50 mg/kg had a 529% effect (Table 3).

Fig. 1.

The effect of Citrus limon (lemon) seed extracts on thiopental-Na stimulated sleeping time in mice (n = 5).

Fig. 2.

The effect of Citrus limon (lemon) seed extracts on thiopental-Na induced latent period on mice (n = 5).

In the hole cross arrangement, the number of holes crossed from one compartment to another was recorded in intervals ranging from 30 to 120 min. It was observed that the seed extracts at 50 and 100 mg/kg doses caused a reduction of activity in the animals (Fig. 3). Robust (p 0.05) data have been obtained in dose-dependent terms (Table 4). In this study, evaluation at the 5th period demonstrated a median of 69% suppression of locomotors activity by seed extract at 100 mg/kg. In the same experimental set-up, diazepam demonstrated 53% suppression of the activity.

Fig. 3.

CNS depressant activity of Citrus limon (lemon) seed extracts by hole cross method (n = 5).

The evaluation of CNS depressant activity by seed extracts provided some interesting results based on the dosage (p 0.05). The extract resulted in significantly minimizing of head-dips in the treated animals in the hole-board test (Fig. 4). The 100 mg/kg dose of seed extract resulted in the suppression of movement (72%), which was stronger than the standard diazepam (67%) (Table 5).

Fig. 4.

CNS depressant activity of Citrus limon (lemon) seed extracts by hole board method (n = 5).

The extracts substantially reduced the locomotor activity in tested mice at 50 and 100 mg/kg doses (p 0.05). This result was noticeable from the underlying perception duration (0 min) and was consistent with the fifth perception duration (120 min) (Fig. 5). From the 2nd inspection to the 5th inspection, diazepam (1 mg/kg) demonstrated a substantial decline in locomotor activity in mice. This test showed a maximum of 36% suppression of locomotor function with seed extract at 100 mg/kg, while 37% was reported with the standard drug diazepam (Table 6).

Fig. 5.

CNS depressant activity of Citrus limon (lemon) seed extracts by open-field method (n = 5).