*Article* **Antidepressant-Like Effect of Traditional Medicinal Plant Carthamus Tinctorius in Mice Model through Neuro-Behavioral Tests and Transcriptomic Approach**

**Mohamed H. Alegiry <sup>1</sup> , Abdelfatteh El Omri 1,2 , Ahmed Atef Bayoumi <sup>1</sup> , Mohammed Y. Alomar <sup>1</sup> , Irfan A. Rather 1,3,\* and Jamal S. M. Sabir 1,3,\***


**Abstract:** Major depression disorder (MDD) has become a common life-threatening disorder. Despite the number of studies and the introduced antidepressants, MDD remains a major global health issue. *Carthamus tinctorius* (safflower) is traditionally used for food and medical purposes. This study investigated the chemical profile and the antidepressant-like effect of the Carthamus tincto-rius hot water extract in male mice and its mechanism using a transcriptomic analysis. The antidepressant effect of hot water extract (50 mg/kg and 150 mg/kg) was investigated in mice versus the untreated group (saline) and positive control group (fluoxetine 10 mg/kg). Hippocampus transcriptome changes were investigated to understand the *Carthamus tinctorius* mechanism of action. The GC-MS analysis of *Carthamus tinctorius* showed that hot water extract yielded the highest amount of oleamide as the most active ingredient. Neuro-behavioral tests demonstrated that the safflower treatment significantly reduced immobility time in TST and FST and improved performance in the YMSAT compared to the control group. RNA-seq analysis revealed a significant differential gene expression pattern in several genes such as Ube2j2, Ncor1, Tuba1c, Grik1, Msmo1, and Casp9 related to MDD regulation in 50 mg/kg safflower treatment as compared to untreated and fluoxetine-treated groups. Our findings demonstrated the antidepressant-like effect of safflower hot water extract and its bioactive ingredient oleamide on mice, validated by a significantly shortened immobility time in TST and FST and an increase in the percentage of spontaneous alternation.

**Keywords:** safflower; oleamide; depression; transcriptome; neuro-behavioral test; grik1; casp9

#### **1. Introduction**

Major depression disorder (MMD) is one of the most common diseases that affect the quality of life of millions worldwide [1]. According to WHO, 264 million people of all ages suffer from depression, and around 850,000 suicides annually [2]. In countries such as the United States, Canada, and China, the lifetime prevalence of MMD varies between 16.2%, 11.3%, and 3.4%, respectively [3]. Besides the modern lifestyle contributing to stress and MDD, the outbreak of COVID-19 had a huge impact on the mental health of many individuals. During the pandemic, the fear of being sick, loss of beloved ones, social distancing, self-isolation, remote education, remote working, loss of job, and dramatic change in daily life activities, among others, negatively impacted our mental health and led to severe psychological distress including depression [4]. In Saudi Arabia, it has been reported that at the beginning of the pandemic, nearly one-fourth of the population experienced psychological impacts that ranged from moderate to severe [5].

**Citation:** Alegiry, M.H.; El Omri, A.; Bayoumi, A.A.; Alomar, M.Y.; Rather, I.A.; Sabir, J.S.M. Antidepressant-Like Effect of Traditional Medicinal Plant Carthamus Tinctorius in Mice Model through Neuro-Behavioral Tests and Transcriptomic Approach. *Appl. Sci.* **2022**, *12*, 5594. https://doi.org/ 10.3390/app12115594

Academic Editors: Luca Mazzoni, Maria Teresa Ariza Fernández and Franco Capocasa

Received: 14 May 2022 Accepted: 30 May 2022 Published: 31 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

MDD is a mental and mood disorder, and it is considered a complex disease. Patients suffering from depression usually suffer from low self-esteem, loss of pleasure, loss of interest, lack of arousal, sleep disturbance, loss of appetite, loss of libido, delusion, hallucinations, and suicidal tentative. Moreover, depression episodes may severely impact a patient's life, social relations, and general health, leading in some cases to life-long disabilities [2,6]. With different causative factors involved, such as heritability and environmental factors, the mechanism of MDD is still unclear, and no mechanism could explain it [7]. Several antidepressants have been developed to treat MDD, including tricyclic antidepressants, monoamine oxidase inhibitors, selective serotonin reuptake inhibitors (SSRIs), Atypical antidepressants, and serotonin-norepinephrine reuptake inhibitors (SNRIs). However, the effect of these drugs seems to be questionable in terms of benefits versus side effects [8]. These therapeutics have not provided the needed relief for those who suffer.

On the contrary, there is a common risk of relapse/recurrence of depressive symptoms after successful acute MDD treatment [1]. Recently, there has been much scientific evidence that complementary and alternative medicine (CAM), which is not considered to be part of conventional medicine, is promising as an alternative to treat and manage mental distress. There are over 120 CAM formulations meant to alleviate and or treat MDD symptoms [9]. A growing body of evidence supports that dietary phytochemicals such as polyphenols (a special class of natural active ingredients) could provide a suitable option to improve mental health alone or integrated with allopathic medicine with minimum side effects [10]. However, more scientific evidence is needed to confirm their activity and understand their molecular mechanisms. In our previous research, we conducted a survey among Saudi people about the ethnobotanical preparations to alleviate stress and depression. We could identify safflower as one of the most cited herbal preparations [11]. Traditionally, safflower petals are soaked in water for around 2 h at room temperature. The filtrate is ingested orally during sorrow, distress, and panic events as a sedative and hypnotic.

Safflower (*Carthamus tinctorius*) is a part of the Composite or Asteraceae family, traditionally used for culinary and medicinal purposes [12]. It was demonstrated to exert numerous medical properties such as antioxidant, analgesic, anti-inflammatory, and antidiabetic [13]. Moreover, safflower has shown anticoagulant, vasodilating, antihypertensive, neuroprotective, and immunosuppressive activities [12].

The present study was conducted based on the ethnopharmacological background to evaluate the antidepressant-like effect of safflower dried petals in mice using different neuro-behavioral tests, optimize the best extraction technique, fingerprint the main active entities, and understand the molecular mechanism using transcriptomic profiling of mice hippocampus.

#### **2. Materials and Methods**

#### *2.1. Preparation of Safflower (Carthamus tinctorius)*

Safflower dried petals (SFP) were crushed in a mortar and extracted at 10 g/100 mL using different techniques: (i) hydro-alcoholic maceration (SFP soaked in 70% ethanol for 24 h, at 10 g/100 mL with continuous shaking), (ii) hot water extraction (extraction at 115 ◦C for 1 min and allowing the maceration to cool down in the autoclave), and (iii) the traditional method used in the Arabic peninsula (Kingdom of Saudi Arabia) which consists of soaking SFP in distilled water for 2, 12, and 24 h at room temperature. The obtained macerate was filtered using the Whatman filter (1001-150, 11 µm particle retention). The filtrate was placed in glass Petri dishes and dried at 37 ◦C in the incubator. Dried resin with dark orange to brown color was then collected in Eppendorf tubes and stored at −80 ◦C for further experiments. For convenience, SFP extracts are abbreviated as follows: 70% ethanol extract: (SFPE), hot water extract: SFPWH, and water extract at room temperature for 2 h (SFPW2), 12 h (SFPW12), and 24 h (SFPW24).

#### *2.2. Safflower Extract Chemical Profiling Using GC-MS*

The GC-MS system (Agilent Technologies, Santa Clara, CA, USA) used in the current study was equipped with a gas chromatograph (7890B) and mass spectrometer detector (5977A) at Central Laboratories Network, National Research Centre, Cairo, Egypt. The GC was equipped with an HP-5MS column (30 m × 0.25 mm internal diameter and 0.25 µm film thickness). Analyses were carried out using helium as a gas carrier at a flow rate of 1 mL/min at a splitless mode, injection volume of 1 µL, and the following temperature program: 60 ◦C for 2 min; rising at 10 ◦C/min to 280 ◦C and held for 10 min. The injector and detector were held at 250 and 300 ◦C, respectively. Mass spectra were obtained by electron ionization (EI) at 70 eV and using a spectral range of *m*/*z* 50–550 and solvent delay of 3 min. Identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.

#### *2.3. Animals*

The animal experiment was conducted in the animal laboratory of the Department of Biological Sciences, Faculty of Sciences at King Abdulaziz University, Jeddah, Saudi Arabia. The study was approved by the ethical committee of the university (201060288). Thirty-two (32) male albino mice were 8 weeks old, with an average body weight of 30.53 g. Animals were housed individually under a 12/12 h light/dark cycle at 25 ◦C, with free access to food and water. Animals were allowed to acclimatize to the laboratory conditions for 7 days prior to the experimentation. All experiments were carried out between 9:00 and 16:00. On the last day of the experiment, mice were sacrificed, and the brain tissue was collected.

#### *2.4. Carthamus tinctorius Administration in Mice*

Fluoxetine and SFPWH were freshly prepared daily in saline prior to the experiments. Drug and extracts were administered by oral gavage using a 1 mL syringe and 20 G × 25 mm (2 mm tip diameter)/straight gavage needle (Petsurgical (AFN2425S)) daily for 15 consecutive days at 9:00 AM, and all mice were administered the same equivalent volumes. Mice were randomly divided into 4 groups (n = 8 each). Control group: administered 200 µL of normal saline per day; positive control group: fluoxetine 10 mg/kg per day; treatment groups 50 and 150 mg/kg SFPWH extract per day.

#### *2.5. Neuro-Behavioral Tests*

Neuro-behavioral tests were conducted in mice 3 h after treatment. The tail suspension test (TST) and forced swimming test (FST) were conducted 7 days each. On the last day of the experiment (day 15), alternation in the Y maze test (YMT) was conducted for each mouse, and animals were sacrificed by spinal cord dislocation, the brain was removed in PBS on ice, and the hippocampus was collected and immediately frozen in liquid nitrogen, then stored at −80 °C for further experiments.

The tail suspension test (TST) has been widely used to evaluate and assess antidepressant activity in mice [14]. Treatment was given 3 h before the beginning of the experiment. Mice from each group were suspended simultaneously, using tape placed at 1 cm from the tip of the tail and hooked to the roof, inside a 4-chamber nonreflective black plexiglass box for 6 min. Animal behavior was recorded using a video camera, and the last 4 min of the experiment were considered to assess TST in SFPWH extract in mice. Recorded videos were analyzed manually, and scores were assigned to each mouse based on our previous study [15].

#### *2.6. Forced Swimming Test (FST) in Mice*

Mice from each group were individually and simultaneously forced to swim in 4 open cylindrical containers (diameter 20 cm, height 30 cm) containing 15 cm of water at 25 ± 1 ◦C. FST sessions duration was 6 min, and the immobility time was recorded during the last

4 min of each experiment. Mice are considered immobile when they remain floating motionless in the water, making only necessary limb movements to keep floating above water [16]. Scores were assigned based on recorded videos.

#### *2.7. Y Maze Test in Mice*

Mice were individually placed in a starting point with a gate at arm A. Exploring time was not allowed. The duration of each test was 10 min, which took place on the last day of the experiment (day 15). The alternation occurred when the mouse moved from one arm and entered entirely into one of the other two arms with its four limbs. The number of alternations between 3 arms was calculated and reported as a percentage of the total number of entries (ABC, ACB, BAC, BCA, CAB, CBA) with no re-entry counted. The way maze was made from black nonreflective plexiglass. It consists of 3 equal arms (length, width, height: 35 × 5 × 10 cm) in a Y shape. Alternation was tracked and scored visually.

#### *2.8. Transcriptomic Analysis (RNA-Seq)*

The hippocampus was dissected from the brain tissue. The total RNA of three mice of each group was extracted using PureLink™ RNA Mini Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The RNA was incubated for 1 h at 37 ◦C with DNase I (ThermoFisher Scientific, Waltham, MA, USA) before heating to 90 ◦C for 30 min. Quantity/quality and RNA integrity were assessed by running the RNA sample on an Agilent Bioanalyzer® RNA 6000 Nano/Pico Chip. mRNA was extracted using NEBNext Poly(A) mRNA Magnetic Isolation beads following the manufacturer's protocol. cDNA libraries were constructed using the NEBNext Ultra II Directional RNA-Seq library. Sequencing was carried out using the Illumina NovaSeq 6000 SP v1.5 Lane (150PE) libraries at the Earlham Institute Genomics core facility. The raw data were processed by the Trinity RNA-seq assembly package [17]. Briefly, the Trimmomatic tool was used in order to remove reads containing adapters. Then, the reads were quantified and mapped to the reference transcript of GRCm39 (Ensembl, Cambridge, UK) by Bowtie v0.12.1. software [18], RSEM v1.1.6 [19] had been used for transcription quantification.

Differential expression analysis of expected read counts was performed by EdgeR (version 3.0.0, R version 2.1.5) [20]. Quantification of transcript expression levels was presented by FPKM (fragments per kilobase of exon per million fragments mapped). The 3rd replicate of SFPWH (150 mg/kg) was excluded due to the lack of consistency.

#### **3. Results**

#### *3.1. Preliminary Phytochemical Screening*

Gas chromatography-mass spectrometry was applied to study the chemical profile of *Carthamus tinctorius* extracts to profile the bioactive ingredients in each extract, including 70% ethanol extraction, water/heat extraction, and traditional extraction. GC-MS analysis showed the presence of oleamide (9-Octadecenamide C18H35NO), as shown in Table 1, which is a fatty amide derived from oleic acid and is considered a potential treatment for mood and sleep disorders [21]. The detailed GC/MS profiling is shown in Table S1. As shown in Figure 1a, hot water extraction yielded the maximum amount of oleamide with 46.52% compared to the other extracts, where 70% ethanol extract yielded 11.15% of oleamide. The traditional extraction method for 2, 12, and 24 h have yielded 34.22%, 32.28%, and 34.53%, respectively. Our results indicate that the maceration time at room temperature did not improve the yield of oleamide. However, hot water extraction at 115 ◦C for 1 min increased the oleamide level by almost 1.5-fold.

#### *3.2. Effect of Safflower Hot Water Extract on Mice Body Weight*

The body weight of each mouse was measured daily before the oral administration session. The data show no significant change in the body weight during the 15-day experiment at *p* < 0.05 (Figure 1b).

**Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.**chemical constituents present *Carthamus* water (detailed to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1). **Table 1.** Major chemical constituents present in *Carthamus tinctorius* hot water extract (detailed profiling refer to Table S1).

mice (the control group), where it fluctuated in the interval [60 s–107 s]. In contrast, safflower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. mice (the control group), where it fluctuated in the interval [60 s–107 s]. In contrast, safflower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. flower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. flower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. flower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. flower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. flower-treated groups and 150 with 10 have shown a the at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respecflower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. flower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively. **Figure 1.** Characterization and efficacy of extracts. (**a**) Oleamide (CH3(CH2)7CH=CH(CH2)7CONH<sup>2</sup> (CID 5283387)) yield in *Carthamus tinctorius* extracts. (SFPE) 70% ethanol for 24 h, (SFPWH) hot water extraction, and (SFPW) the traditional method using maceration in water at room temperature for 2, 12, and 24 h. Oleamide (%) is reported as the percentage of the sum area in each extract. (**b**) Daily effect of safflower hot water extract treatment on body weight in depression-induced mice. Data represent the mean ± SEM (n = 8 mice). **Figure 1.** Characterization and efficacy of extracts. (**a**) Oleamide (CH<sup>3</sup> (CH<sup>2</sup> )7CH=CH(CH<sup>2</sup> )7CONH<sup>2</sup> (CID 5283387)) yield in *Carthamus tinctorius* extracts. (SFPE) 70% ethanol for 24 h, (SFPWH) hot water extraction, and (SFPW) the traditional method using maceration in water at room temperature for 2, 12, and 24 h. Oleamide (%) is reported as the percentage of the sum area in each extract. (**b**) Daily effect of safflower hot water extract treatment on body weight in depression-induced mice. Data represent the mean ± SEM (n = 8 mice).

**Figure 2.** Effect of safflower hot water extract on immobility time in TST. (**a**) Effect on overall immobility time in TST-induced stress in mice. (**b**). Effect on overall immobility time in FST-induced stress in mice. ns = non-significant, \* and \*\* indicates significantly different at p < 0.05 and *p* < 0.01,

Mice from each group were individually and simultaneously forced to swim in 4 open cylindrical containers (diameter 20 cm, height 30 cm) containing 15 cm of water at 25 ± 1 °C (Figure 2B). FST sessions duration was 6 min, and the immobility time was recorded during the last 4 min of each experiment. Mice are considered immobile when they

mice (the control group), where it fluctuated in the interval [60 s–107 s]. In contrast, saf-

mice (the control group), where it fluctuated in the interval [60 s–107 s]. In contrast, saf-

mice (the control group), where it fluctuated in the interval [60 s–107 s]. In contrast, saf-

respectively versus untreated group, Student t-test.

*2.6. Forced Swimming Test (FST) in Mice*

#### *3.3. Tail Suspension Test (TST)*

The antidepressant-like effect of *Carthamus tinctorius* hot water extract was evaluated every other day using a tail suspension test in mice fed with the extract for 14 days. As shown in Figure 2a, a significant reduction in the immobility time during the last 4 min of the total 6 min period tail suspension was noticed (*p* < 0.05) in fluoxetine 10 mg/kg (47.921 ± 8.725 s) and safflower hot water extract treatments at 50 mg/kg (45.398 ± 5.564 s) and 150 mg/kg (51.940 ± 3.506 s) as compared to the control group (84.375 ± 10.513 s). **Figure 1.** Characterization and efficacy of extracts. (**a**) Oleamide (CH3(CH2)7CH=CH(CH2)7CONH<sup>2</sup> (CID 5283387)) yield in *Carthamus tinctorius* extracts. (SFPE) 70% ethanol for 24 h, (SFPWH) hot water extraction, and (SFPW) the traditional method using maceration in water at room temperature for 2, 12, and 24 h. Oleamide (%) is reported as the percentage of the sum area in each extract. (**b**) Daily effect of safflower hot water extract treatment on body weight in depression-induced mice. Data represent the mean ± SEM (n = 8 mice).

*Appl. Sci.* **2022**, *12*, x FOR PEER REVIEW 4 of 14

**Figure 2.** Effect of safflower hot water extract on immobility time in TST. (**a**) Effect on overall immobility time in TST-induced stress in mice. (**b**). Effect on overall immobility time in FST-induced stress in mice. ns = non-significant, \* and \*\* indicates significantly different at p < 0.05 and *p* < 0.01, respectively versus untreated group, Student t-test. **Figure 2.** Effect of safflower hot water extract on immobility time in TST. (**a**) Effect on overall immobility time in TST-induced stress in mice. (**b**). Effect on overall immobility time in FST-induced stress in mice. ns = non-significant, \* and \*\* indicates significantly different at *p* < 0.05 and *p* < 0.01, respectively versus untreated group, Student t-test.

*2.6. Forced Swimming Test (FST) in Mice* Mice from each group were individually and simultaneously forced to swim in 4 open cylindrical containers (diameter 20 cm, height 30 cm) containing 15 cm of water at 25 ± 1 °C (Figure 2B). FST sessions duration was 6 min, and the immobility time was recorded during the last 4 min of each experiment. Mice are considered immobile when they The immobility time in all mice groups was wavy with a general trend of increase. The TST immobility time increase was the highest in the untreated mice (the control group), where it fluctuated in the interval [60 s–107 s]. In contrast, safflower-treated groups (50 and 150 mg/kg) along with positive control (Fluoxetine 10 mg/kg) group have shown a significant reduction in the immobility times at the same period fluctuating the following intervals [31 s–56 s], [12 s–64 s], and [19 s–54 s], respectively.

#### *3.4. Forced Swimming Test (FST)*

Safflower hot water extract-treated mice were subjected to FST compared to the untreated group (vehicle) and fluoxetine-treated mice as a positive control group. The antidepressant effect was assessed based on the last 4 min of 6 min session immobility time in FST. Mice were considered immobile when they were motionless, floating above the water. As indicated in Figure 2b, safflower and fluoxetine showed a mild effect on immobility time in FST. The maximum reduction in the immobility time was observed in SFPWH (150 mg/kg) with 80.601 ± 13.52 s compared to the control group 101.949 ± 19.317 s and fluoxetine (10 mg/kg) group 100.501 ± 16.556 s.

#### *3.5. Y Maze Spontaneous Alternation Test (YMSAT)*

YMSAT is a behavioral test used to evaluate the exploratory behavior in mice or rodents in general. Typically, rodents in normal conditions prefer to explore a new arm instead of re-visiting the same arm they explored before. This exercise involves several brain parts, including the hippocampus and prefrontal cortex [22]. YMSAT is commonly used to assess and quantify cognitive deficits in rodents exposed to novel chemical entities [23].

As indicated in Figure 3, mice treated with fluoxetine and safflower hot water extract 50 mg/kg and 150 mg/kg had their exploratory behavior significantly improved and scored 68.41 ± 3.16%, 67.12 ± 1.39%, and 72.82 ± 2.08%, respectively in the percentage of spontaneous alternation as compared to stressed and untreated mice (vehicle group) (60.41 ± 0.98%). Results indicated better performance in safflower-treated mice than in the fluoxetine-treated group. As indicated in Figure 3, mice treated with fluoxetine and safflower hot water extract 50 mg/kg and 150 mg/kg had their exploratory behavior significantly improved and scored 68.41 ± 3.16%, 67.12 ± 1.39%, and 72.82 ± 2.08%, respectively in the percentage of spontaneous alternation as compared to stressed and untreated mice (vehicle group) (60.41 ± 0.98%). Results indicated better performance in safflower-treated mice than in the fluoxetine-treated group.

Safflower hot water extract-treated mice were subjected to FST compared to the untreated group (vehicle) and fluoxetine-treated mice as a positive control group. The antidepressant effect was assessed based on the last 4 min of 6 min session immobility time in FST. Mice were considered immobile when they were motionless, floating above the water. As indicated in Figure 2c, safflower and fluoxetine showed a mild effect on immobility time in FST. The maximum reduction in the immobility time was observed in SFPWH (150 mg/kg) with 80.601 ± 13.52 s compared to the control group 101.949 ± 19.317

YMSAT is a behavioral test used to evaluate the exploratory behavior in mice or rodents in general. Typically, rodents in normal conditions prefer to explore a new arm instead of re-visiting the same arm they explored before. This exercise involves several brain parts, including the hippocampus and prefrontal cortex [22]. YMSAT is commonly used to assess and quantify cognitive deficits in rodents exposed to novel chemical entities [23].

*Appl. Sci.* **2022**, *12*, x FOR PEER REVIEW 7 of 14

s and fluoxetine (10 mg/kg) group 100.501 ± 16.556 s.

*3.5. Y Maze Spontaneous Alternation Test (YMSAT)*

*3.4. Forced Swimming Test (FST)*

**Figure 3.** Effect of safflower water extract treatment on spontaneous alternation in YMT. Each mouse was allowed to explore the Y maze in an 8 min session. The number of alternations between 3 arms was calculated and reported as a percentage of the total number of entries. Each bar represents the mean ± SEM (n = 8). \* and \*\* indicates significantly different at *p* < 0.05 versus untreated group, Student *t*-test. **Figure 3.** Effect of safflower water extract treatment on spontaneous alternation in YMT. Each mouse was allowed to explore the Y maze in an 8 min session. The number of alternations between 3 arms was calculated and reported as a percentage of the total number of entries. Each bar represents the mean ± SEM (n = 8). \* and \*\* indicates significantly different at *p* < 0.05 versus untreated group, Student *t*-test.

#### *3.6. Transcriptomic Analysis (RNA-Seq) 3.6. Transcriptomic Analysis (RNA-Seq)*

Using next-generation sequencing, RNA-seq is a powerful tool capable of detecting the quantitative measurement of RNA abundance or gene expression [24]. RNA-seq analysis of the hippocampus tissue was used to compare the differential gene expression pattern of the vehicle group compared to SFPWH-treated and fluoxetine-treated groups. As Using next-generation sequencing, RNA-seq is a powerful tool capable of detecting the quantitative measurement of RNA abundance or gene expression [24]. RNA-seq analysis of the hippocampus tissue was used to compare the differential gene expression pattern of the vehicle group compared to SFPWH-treated and fluoxetine-treated groups. As shown in Figure 4a, 72 detected subclusters and 82 transcripts. Gene ontology enrichment analysis (Figure 4b) was performed by ShinyGO v0.741 [25], revealing the top pathways for the detected genes. Data analysis showed a significant differential gene expression pattern in 22 genes, which might be potentially related to mood disorders (Figure 4c).

The expression pattern of the mean SFPWH (50 mg/kg) group replicates revealed a significantly different gene expression compared to the control group. Where 11 transcripts were downregulated in genes (Ube2j2, Dctn6, Ncor1, Acp2, Tuba1c, Gm14150, Actr3-ps, Grik1, Commd5, and Gm3226). In addition, 7 transcripts were upregulated in genes (Msmo1, Casp9, Hspe1-rs1, Tmem44, Rps13-ps2, Rps3a2,and Dctn6). In comparison to the control group. At the same time, fluoxetine-treated group showed a significantly different gene expression in six genes where two transcripts were downregulated in genes Cacna1h and Dync1i2. Moreover, five transcripts were upregulated in genes (Rbl2, Mas1, Hyi, Morf4l2, and Dync1i2). SFPWH (150 mg/kg) group has not shown any significant gene expression compared to the control group (Table 2).

gene expression compared to the control group (Table 2).

different gene expression in six genes where two transcripts were downregulated in genes Cacna1h and Dync1i2. Moreover, five transcripts were upregulated in genes (Rbl2, Mas1, Hyi, Morf4l2, and Dync1i2). SFPWH (150 mg/kg) group has not shown any significant

**Figure 4.** Transcriptome differences detected between the mean of the control group and treatment groups. (**a**). Hierarchical cluster analysis of gene expression based on log-ratio FPKM data for the mean of transcripts replicates illustrating the impact of the administration of safflower hot water extract and fluoxetine treatment in comparison to control where red indicates upregulation pattern while blue color indicates downregulation pattern. (**b**). Heatmap showing significantly different gene expression patterns (log2 fold). (**c**). The bar charts for the gene ontology enrichment analysis **Figure 4.** Transcriptome differences detected between the mean of the control group and treatment groups. (**a**). Hierarchical cluster analysis of gene expression based on log-ratio FPKM data for the mean of transcripts replicates illustrating the impact of the administration of safflower hot water extract and fluoxetine treatment in comparison to control where red indicates upregulation pattern while blue color indicates downregulation pattern. (**b**). Heatmap showing significantly different gene expression patterns (log2 fold). (**c**). The bar charts for the gene ontology enrichment analysis show the top pathways for the significant enrichment genes for all groups. *p*-value cutoff (FDR) 0.05.

**Table 2.** List of the significant differential expressed genes of safflower hot water extract SFPWH **Table 2.** List of the significant differential expressed genes of safflower hot water extract SFPWH (50 mg/kg) and fluoxetine-treated group compared to the control group.

show the top pathways for the significant enrichment genes for all groups. *p*-value cutoff (FDR) 0.05.


*Dctn6-202* 22,428 1.84

#### **4. Discussion**

In recent years, dietary phytochemicals and botanicals received the scientific community's attention regarding their potential use as a complementary and alternative therapy to either prevent, manage, or treat several diseases, including stress and depressive symptoms, with minimum side effects, in comparison to allopathic treatments, known to exert several adverse effects such as gastrointestinal problems, sleep disturbances, sexual dysfunction, congenital disabilities, seizures, and even death, among others [8]. Dietary phytochemicals are a part of our daily diet, easy to find, and cheap. Additionally, they have a safe "green image" that may potentially help to prevent and alleviate MDD symptoms. In this respect, many studies and experiments demonstrated that several polyphenols might produce an antidepressant-like effect [26]. The Chinese Health Ministry has recorded safflower formulations as a drug against cardiovascular diseases [27]. Safflower is one of the plants that has been used in traditional and modern medicine for years in several countries. Many studies illustrated the protective effect and other health benefits on musculoskeletal and cardiovascular systems and reproductive organs [12]. Safflower is known for its bioactivities such as anti-inflammatory, antioxidant, and antitumor effects [28]. Precisely, the water extract of safflower is considered an anticoagulant, vasodilating, antihypertensive, antioxidative, neuroprotective, immunosuppressive, and anticancer agent [12]. Our study demonstrated that safflower dried petals hot water extract exerts an antidepressant effect in mice subjected to TST and FST-induced stress, as shown by the improvement in the percentage of spontaneous alternation in YMSAT. The tail suspension test is an inexpensive and easy-to-use behavioral test used to detect depression-related behaviors and measure the potentiality of antidepressants on mice since the 1980s. TST has advantages over other behavioral tests in overcoming motor dysfunction of the mice and hypothermia issues [29].

A forced swimming test is also another widely used, easy, inexpensive, and fast behavioral test that could be used along with TST. TST and FST both use immobility as a measurement that represents depressive behavior. FST contributes to depression research, especially in genetic analysis, where immobility in FST is determined by heritable traits [30]. The Y maze test could easily assess the memory and learning patterns of mice, which allows continuous spontaneous alternation. During this test, the hippocampus is hugely involved in retrieving essential information for building a mental map [31]. Fluoxetine (C17H18F3NO) is a high CNS penetration, orally administrated, and widely used drug, which is a selective serotonin reuptake inhibitor (SSRI) antidepressant [32].

Chromatographic analysis showed that oleamide (9-octadecenamide) would be the main active ingredient extracted in high yield in water than in hydro-alcoholic solvent. Moreover, we report for the first time the optimum procedure of extraction that yielded 46% oleamide-rich tea by soaking safflower in water at 115 ◦C for 1 min. Olamide is known as a hypnotic and sedative chemical entity that naturally accumulates in human and mammals cerebrospinal fluid during sleeping deprivation as a natural modulator to induce sleeping and/or to reduce psychological excitement through the interaction with the cannabinoid receptor [33]. Additionally, oleamide was reported to interact with several neurotransmitter-receptor systems, including serotonin and GABA [34]. Moreover, oleamide provides anti-inflammatory activity along with other pharmacological properties such as human monoamine oxidase B enzyme inhibitor and TRPV1 vanilloid receptors activator [35].

Our study reports for the first-time safflower extract-administered mice 'hippocampus transcriptomic profiling. Transcriptome analysis, including highly reproducible and significant target genes (*p* < 0.001), resulted in a pool of 22 genes. Interestingly, some of the genes are highly relevant to MDD, anxiety, and neurobehavior. A study on rats has revealed that NcoR (nuclear receptor corepressor) expression reduction within the developing amygdala is associated with a significant increase in anxiety-like behavior during the juvenile period in both genders [36]. On the contrary, hippocampal NcoR1 was upregulated in the control group with an average of 1.33 log2 for the three replicates, while a significant reduction in expression level was detected in SFPWH (50 mg/kg) group with −1.4 log2 fold. However, no significant difference was detected in SFPWH (150 mg/kg) and fluoxetine (10 mg/kg) groups. The gene ontology enrichment analysis showed that NcoR1 involves in the thyroid hormone signaling pathway (33.15-fold enrichment, FDR 0.0030). Noting that an adequate level of thyroid hormone is essential for the brain to function normally [37].

Apoptosis plays an important role in the development of the brain and the peripheral nervous system as well [38]. There is a link between apoptosis and major depressive disorder and other neurodegenerative diseases such as Parkinson's and Alzheimer's disease [38]. Casp9 is one of the differentially expressed genes in the SFPWH (50 mg/kg)-treated group and the enrichment analysis revealed involvement in several pathways, including apoptosis and neurodegeneration. Interestingly, casp9 was highly expressed in SFPWH (50 mg/kg) group (2.67 log2 fold) in comparison with the control group (−1.27 log2 fold).

Glutamate is a neurotransmitter that has an excitatory effect [39]. Glutamate system abnormalities and behavior that lead to several disorders, including MDD, were linked [39]. In our study, Grik1 (glutamate receptor, ionotropic, kainate 1) was significantly downregulated (−1.29 log2 fold) in SFPWH (50 mg/kg) group. It has been reported that female patients with MDD showed a significantly higher expression level of the Grik1 gene in the dorsolateral prefrontal cortex [39].

Ube2j2 gene is predicted to be involved in protein polyubiquitination and ubiquitindependent endoplasmic reticulum-associated protein degradation pathway [40]. Endoplasmic reticulum stress has a major role in the pathophysiology of depression, where an abnormal function is linked with triggering apoptosis signals [41]. In our study, Ube2j2 was significantly downregulated in SFPWH (50 mg/kg) group (−2.06 log2 fold) compared to the control group.

Transcriptomic analysis revealed two transcripts of the Dctn6 gene that were expressed differently. However, Dctn6-202 is the transcript that encodes the protein Dynactin subunit 6. The function of Dctn6 involves enabling dynein complex binding activity [42]. It has been reported that chronic stress reduces the dynein motor protein expression in the rat hippocampus [43]. In SFPWH (50 mg/kg) group, the Dctn6-206 transcript was downregulated (−1.90 log2 fold) while the transcript Dctn6-202 was upregulated (1.84 log2 fold). Tuba1c is another significantly downregulated gene detected in SFPWH (50 mg/kg) group. It is predicted that Tuba1c, which encodes Tubulin alpha-1C chain protein, enables GTP binding activity and structural constituent of the cytoskeleton [44]. Cytoskeletal (microtubules) post-translation modification is linked with several neuropsychiatric diseases [45]. In our study, the data analysis showed that Tuba1c is involved with several pathways, including neurodegeneration, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and apoptosis pathways. Cacna1h gene was significantly downregulated in the fluoxetine-treated group with an average of −1.42 log2 fold. It has been reported that mice deficient in CaV3.2 channels, which is encoded by the Cacna1h gene, showed an increase in anxiety-like behavior [46], where safflower hot water extract-treated and control groups were not significantly different.

#### **5. Conclusions**

Our results represent the first scientific evidence supporting the ethnopharmacological use of safflower and its main active ingredient, oleamide, as a hypnotic and sedative. These properties are mainly related to a large amount of oleamide in the hot water extract. To the best of our knowledge, our study is the first study to elucidate the mechanism of action of safflower through the regulation of several genes related to MDD using a transcriptomic approach. Further experiments are needed to validate the RNA-seq outcomes at translational and post-translational levels. Our study may provide insight into the mechanism of action of safflower extract that may interfere with the apoptosis, glutamate, and endoplasmic reticulum-associated protein degradation pathways playing essential roles in protection against depression. Further studies on this issue at the post-translational level using the transcriptomic approach and immuno-histochemistry targeting different brain parts will

provide more information to understand the mechanism of action of safflower and its main active ingredient, oleamide, in the prevention of depression and stress.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/app12115594/s1, Table S1: Chemical profile of *Carthamus tinctorius* extract.

**Author Contributions:** Conceptualization, M.H.A., A.E.O. and J.S.M.S.; methodology, M.H.A., A.E.O., A.A.B. and M.Y.A.; software, I.A.R.; validation, A.E.O., A.A.B., M.Y.A., I.A.R. and J.S.M.S.; formal analysis, all authors; investigation, M.H.A.; resources, J.S.M.S.; data curation, all authors; writing—original draft preparation, M.H.A.; writing—review and editing, all authors; visualization, all authors; supervision, J.S.M.S.; project administration, A.E.O. and J.S.M.S.; funding acquisition, J.S.M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The animal study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committees, Department of Biological Sciences, King Abdulaziz University, Saudi Arabia (201060288).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The date generated is cited in the manuscript and provided as Supplement File.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

