, Muniruddin Ahmed 1,*1 Department of Pharmacy, Faculty of Allied Health Sciences, Daffodil International University, 1207 Dhaka, Bangladesh
2 Pre-Clinical Research Unit, King Fahd Medical Research Center, King Abdulaziz University, 21589 Jeddah, Saudi Arabia
3 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, 21589 Jeddah, Saudi Arabia
4 Prince Fahd Research Chair, Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, University of Tabuk, 47311 Tabuk, Saudi Arabia
Abstract
Citrus limon L. is an ingenious alternative medication and has a broad scope in managing several health conditions as part of natural remedies. Recently, medicinal plants have witnessed incredible consideration worldwide in the field of neuroscience for remedial intervention. The present work has investigated the phytochemical compounds and neuropharmacological potential of the seed extract of Citrus limon as a step to partially validate its formulations as nutraceuticals using an in vivo model. Diverse phytochemical groups such as alkaloids, glycosides, flavonoids, tannins, gums, saponins, steroids were qualitatively identified through colorimetric methods utilizing standard compounds. The neuropharmacological properties were studied in Swiss albino mice with the sleep time induced by thiopental sodium taken as an end-point, in standard hole cross, hole board, and open-field experiments at varying doses of 50 and 100 mg/kg body weight. Phytochemical screening showed that alkaloids, flavonoids, saponins, tannins, steroids, and glycosides are present in the aqueous extract of the seed. The extracts demonstrated a significant reduction in sleep onset and enhanced the sleep duration in a dose-dependent manner in thiopental sodium-induced sleeping time, along with a marked decrease in unconstrained locomotors and explorative properties in both hole cross and open field tests. Moreover, in the hole board study, the extracts minimized the count of head dips observed in the treated mice. The results shown in this study demonstrate that Citrus limon extracts have neuropharmacological properties that can be further examined for their potential role as an adjuvant with conventional medications or nutraceuticals.
Keywords
- Neurological disorders
- Phytochemical compounds
- Latent period
- Neuropharmacological potential
- Nutraceuticals
Recently, a stressed lifestyle has been linked to an assortment of psychiatric disorders, and evidence suggests that depression is an unavoidable medical concern in these disorders [1, 2, 3, 4]. Different antipsychotic drugs, namely, tricyclic antidepressants, monoamine oxidase inhibitors, and serotonin re-uptake inhibitors, are available to manage various psychotic problems, but several side effects are gradually hindering their use in a long-term therapeutic regimen [5, 6]. Thus, the quest for new anxiolytic substances with minimal unfavorable impact is still an area of interest being explored for an appropriate pharmacological effect. Certain organic substances, particularly restorative herbs, are commonly known to be the primary armamentarium of concoction compounds with enormous remedial potential. Neuropsychiatric disorders such as depression are common, corresponding to a state of mental illness that may result in severe symptoms such as sadness, feelings of guilt and loss of interest which affect sleeping, dietary intake, cognitive and psychomotor guided daily activities. Depression and anxiety, if they remain untreated, may aggravate the symptoms, worsen the complications, and ultimately lead to suicidal ideation risks [7, 8]. The actual etiology of depression and anxiety is still unknown. However, these may arise due to continued disruption of the antioxidant defense system and an enhanced redox imbalance or oxidative stress [9]. The redox stress in the brain supports cellular conditions that cause neurological damage and related cognitive deteriorations [10, 11, 12, 13].
Citrus limon belongs to the Rutaceae family, with a predominant
presence in South Asia, including Northeastern India. Citrus limon is
the most notable citrus species, with an affluent mercantile value. Most types of
citrus plants are substantial evergreen bushes or small trees, 5–15 meters tall.
The tree’s ellipsoidal yellow fruit is used worldwide for culinary and
non-culinary purposes and is an active ingredient of folk medicine and functional
foods. The lemon juice is around 5 to 6 percent citrus extract, having an acrid
flavor with a pH of about 2.2. The distinctively harsh flavor of lemon juice
makes it an essential constituent of drinks and nutritional supplements,
including lemonade and lemon meringue pie. Its berry is essentially rich in
alkaloids which show anticancer properties and demonstrates antibacterial
activities in unrefined concentrations of lemon on clinically challenging
bacterial strains [14, 15, 16, 17, 18, 19, 20, 21]. The biological activity of C. limon is
attributed to the rich quantity of phenolic compounds, for instance, flavonoids;
limonoids, carotenoids and bioactive monoterpenoids such as D-limonene,
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
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
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
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:
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
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
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
Results are shown as mean
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.
| Compounds | Seed (aqueous extracts) |
| Alkaloids | ++ |
| Flavonoids | + |
| Saponins | - |
| Tannins | + |
| Steroids | ++ |
| Gums | + |
| Cardiac glycosides | + |
| All tests were carried out in triplicates. KEY: ++, Highly detected; +, Less detected; -, Not detected. | |
| Phytochemical class | Quantity (%) |
| Alkaloids | 0.36 |
| Flavonoids | 0.34 |
| Tannins | 12.14 |
| The values are represented as the Mean | |
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
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).
| Group | Dose (mg/kg) | Latent period (min) | Duration of sleep (min) | % Effect |
| Control | 10 mL/kg | 9.6 |
37.4 |
0 |
| Standard (Diazepam) | 0.50 | 2.6 |
198.2 |
529.94 |
| Seed extract | 25 | 8.5 |
39.8 |
106.41 |
| Seed extract | 50 | 6.00 |
74.8 |
200.00 |
| Seed extract | 100 | 3.5 |
140.8 |
376.47 |
| Seed extract | 200 | 1.95 |
263.6 |
704.81 |
| Values are represented as Mean | ||||
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
Fig. 3.CNS depressant activity of Citrus limon (lemon) seed extracts by hole cross method (n = 5).
| Group | Dose (mg/kg) | Quantity of moves (% of movements Inhibition) | ||||
| 0 min | 30 min | 60 min | 90 min | 120 min | ||
| Control | 10 mL/kg | 5.0 |
6.2 |
6.2 |
6.6 |
8.6 |
| Standard | 1 | 3.4 |
5.0 |
4.0 |
3.6 |
4.0 |
| Seed extract | 50 | 2.8 |
4.2 |
4.4 |
3.4 |
2.8 |
| Seed extract | 100 | 1.4 |
2.6 |
2.2 |
3.0 |
2.6 |
| Values are represented as Mean | ||||||
The evaluation of CNS depressant activity by seed extracts provided some
interesting results based on the dosage (p
Fig. 4.CNS depressant activity of Citrus limon (lemon) seed extracts by hole board method (n = 5).
| Group | Dose (mg/kg) | Quantity of head dips | % Inhibition |
| Control | 10 mL/kg | 21.4 |
0 |
| Standard | 1 | 7 |
67.28% |
| Seed extract | 50 | 8.4 |
60.74% |
| Seed extract | 100 | 5.8 |
72.89% |
| Values are represented as Mean | |||
The extracts substantially reduced the locomotor activity in tested mice at 50
and 100 mg/kg doses (p
Fig. 5.CNS depressant activity of Citrus limon (lemon) seed extracts by open-field method (n = 5).
| Group | Dose (mg/kg) | Quantity of moves (% of movements Inhibition) | ||||
| 0 min | 30 min | 60 min | 90 min | 120 min | ||
| Control | 10 mL/kg | 26.8 |
28.0 |
28.6 |
31.8 |
38.6 |
| Standard | 1 | 16.2 |
19.2 |
21.8 |
23.0 |
24.2 |
| Seed extract | 50 | 19.4 |
25.2 |
24.2 |
23.0 |
25.4 |
| Seed extract | 100 | 16.6 |
20.4 |
19.2 |
21.6 |
24.4 |
| Values are represented as Mean | ||||||
Traditional medicines have been considered an original form of therapeutic
approach that has been inexpensive and effective since ancient times. Recent
efforts have focused on including neurobehavioral function and efficiency to
complement modern medication with different ethnomedicinal products. Sedative
properties for such bioactive compounds and natural products have been explored
with standard protocols like hole-cross and free field experiments that can
evaluate and record the naturalistic locomotor behavior of mice or its alteration
under induced conditions. Our findings indicate that the oral administration of
the experimental plant seed extract at the doses (50 and 100 mg/kg) resulted in a
significant decrease in the count of holes passed (Table 4). The repressive
behavior was shown at 30 min when the extracts were given and persisted for 120
min. The extracts at the studied doses induced a substantial locomotion reduction
that was observed in the open field test from 30 min to 120 min during the
inspected duration (Table 6). It is known that by observing the total distance,
the open field test can be used to evaluate the anxiety behavior. In the current
study, the resting time’s range was between 6–12 seconds, the duration of time
spent in center squares was 3 min, and the distance traveled into the center
squares was between the ranges of 8.5 m
Moreover, the seed extracts reduced the sleep latency in the thiopental
sodium-mediated sleeping time test (Fig. 2) and prolonged the sleep periods (Fig. 1)
depending on the doses and demonstrated substantial sedative effects. Both
tests suggestively diminished locomotion in all Swiss albino mice treated with
the standard and seed extracts. Animal head-dipping behavior remains closely
correlated with their psychological response. Gamma amino-butyric acid (GABA) can
modulate the central nervous system through the voltage-gated Ca
Recent studies in literature have reported the pharmacological benefits of the lemon constituents on the neurological well-being [68, 69, 70].The current study provides evidence of the pharmacological properties of Citrus limon (lemon) seed extract in complementing the effects of thiopental sodium by restricting the locomotor activity in the mice model. Thiopental sodium is a standard drug for the induction of anaesthesia and hypnosis. The reduction in the sleep onset and an enhanced sleep duration induced in this drug model by the lemon seed extract provide a potential adjuvant for the qualitative augmentation of the existing protocols. The decrease in unconstrained locomotors and explorative properties further demonstrates the anxiolytic potential of the extract. However, further studies are warranted to investigate the plausible modes of action responsible for such neuropharmacological effects of Citrus limon (lemon) seed extract. The study broadens the scope of the lemon fruit as a nutraceutical or functional food with neuro-modulatory pharmacological properties.
C. limon,Citrus limon; Ltd, limited; BDH, British Drug House; HCL, Hydrochloric acid, Normality; M, Molarity; b.w, body weight; CNS, Central nervous system; GABA, Gamma amino-butyric acid.
MMR—Experimental work; FI—Experimental work; AP: Experimental work; MAKA—Statistical analysis; GMA— Conceptualization and Analysis; MFU—Study Design, Analysis and manuscript writing; MA—Conceptualization, Study design, Analysis and Manuscript writing.
Animals were obtained from Jahangirnagar University, Dhaka, Bangladesh. 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).
The authors are grateful to the Department of Pharmacy, Jahangirnagar University, Dhaka, Bangladesh for providing experimental animals. The authors also condole the sudden demise of Dr. Sharif Mohammad Shaheen and gratefully acknowledge his contribution to this work.
This work was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, Saudi Arabia under grant no. (KEP-1-141-41). The authors, therefore, acknowledge with thanks DSR technical and financial support.
The authors declare no conflict of interest.