Antidepressant and Anxiolytic Profiles of Cymbopogon Flexuosus Ethanolic Extract in Chronic Unpredictable Mild Stress Induced in Rats

1Department of Pharmacology and Toxicology, Pharmacy College, Jazan University-KSA. 2Pharmacy College, Jazan University-KSA. 3Departmentof Pharmaceutical Chemistry, College of Pharmacy, Jazan University, Jazan, KSA. 4Department of Pharmacognosy –Pharmacy College-Jazan University-KSA. 5Department of PharmacologyNational Research Centre-Dokki-Giza-Egypt. *Corresponding Author E-mail: dr_g.asaad@yahoo.com

Chronic mild stress model is an approved method for inducing stress in experimental animals 1 . It has been reported that exposure to scheduled unpredictable stressors induces substantial impairment in behavioral status as locomotive and explorative behavior. chronic mild stress model has also been used effectively as an animal model of depression associated with anxiety 2 .
Many studies used rats to determine the effect of many therapeutic agents such as fluoxetine (FLX), on behavioral impairment viz depression associated with anxiety using elevated plus maze, forced swim test. Administration of fluoxetine in an acute or chronic manner was recorded to increase motor activity in familiar environment but enhancement of exploratory activity in the open field was exerted only with repeated administration 3 . A major disadvantage of SSRI treatment is the delayed antidepressant response which is believed to be due to the time needed for improvement of serotonergic neurotransmission. This improvement is thought to be due to the desensitization of somatodendritic 5-HT1A autoreceptors 4 .
Cymbopogon flexusousis placed in the genus Cymbopogon. The essential oil of C. flexusous has shown an anti-inflammatory effect by inhibiting various inflammatory and immunomodulatory biomarkers 5 . C.flexusous also showed an inhibitory effect on the early phase hepatocarcinogenesis in rats after initiation with diethylnitrosamine 6 . The Methanolic extract of C.citratus leaves; one of the Cymbopogon family increased the percentage of time-spent and the percentage of arm entries in the open arms and decreased the percentage of timespent in the closed arms of the elevated plus-maze, and this effect was attributed to the high content of flavonoids in the methanolic extract 7 .
Nowadays, developed countries are investing in traditional herbal medicine research to overcome the detrimental adverse effects and drawbacks of synthetic agents. As examples for these drawbacks; the delayed response and the need of repeated administration of SSRI which may lead to incidence of toxicity. The current study was conducted to assess the antidepressant and anxiolytic activity of C.flexusous ethanolic extract against chronic mild stress model in rats in refer to fluoxetine.

Materials and Methods
Preparation of leave extract of C.flexuosus C.flexuosus leaves were collected from Jazan botanical garden. Identified by the botanist from Jazan University, Pharmacy College and the specimen was kept in the campus. Crude drug (leaves) was left for drying under shade,grinded coarsely by using a mortar and pestle. About 25 gm of powder was weighed, processed for hot percolation method with various solvents like ethyl alcohol, Ethyl acetate, Chloroform depending on their polarity level. Before starting, the process packing was carefully done by placing cotton bed on the upper and lower level of the powder, to avoid any blockage. Temperature of the solvents was adjusted to its boiling point in a heating mantle. Cool water supply was provided for condensation process. Extraction process was stopped after16 hours. Extracts were condensed to remove the solvents and stored for further studies. Solvents and Fluoxetine hydrochloride procured from Sigma Aldrich, Germany.

Preliminary phytochemical screening
Preliminary studies were done to identify the different chemical constituents like alkaloids, flavonoids, steroids, tannins, glycosides, triterpenoids and saponins 8 .

Physiochemical properties used for identification of C.flexuosus GC/Ms analysis
The ethanolic extract of dried leaves of C.flexuosus was subjected to GCMS analysis using Thermo Scientific GC/MS equipped with AS 3000 auto samplers, trace ultra-GC and ISQ detector. The separation was achieved using capillary TR-5MS column (30 m × 0.25 mm ID × 0.25 ìm) and helium carrier gas at a flow rate of 1.2 mL/min using our earlier reported method of sesame oil (9). Ethanolic extract (1mL) was diluted to 10 mL v/v with absolute ethanol, sonicated for 2 minutes, filtered through 0.45ìm filter and injected in split less mode (2 µL). The injection port, ion source and MS line transfer temperatures were set at 320 o C, 320 o C and 340 o C respectively. The oven temperature was initially set at 70 o C with holding time for 5 minutes and subsequently ramped to 205 o C, 280 o C, 290 o C and 300 o C at rate of 5 o C/min with 5 min holding time at ramping stage. For GC-MS detection, an electron ionization system with ionization energy of 70 eV with 0.6 s scan time was used. The spectra were recorded using positive ion mode within the mass range of 20-800 amu. A delay time of 4 min was selected to avoid initial solvent peaks. The mass spectra were interpreted using Xcalibur software and the fragmentation patterns of mass spectra were compared with database using the built-in NIST library. The relative percentage amount of each component was calculated by comparing its average peak area to the total area.

Compound identification
The components ethanolic extract of dried leaves of C.flexuosus were identified by using match factor (SI) and reverse match factor (RSI) thresholds of 900 and above between measured spectrum and standard library spectrum. Percentage areas of each component were obtained by using Xcalibur software without use of any internal standard and are uncorrected. experimental protocol animals Forty-two adult female wistar rats (150-180g) were obtained from the animal breeding unit at the Medical College-Jazan University. Animals were kept in a cleansed, clear poly propylene cages in groups of four in each cage maintained at 25 ± 2ºC with 12 hours light and dark cycle with free access to cubes of food and water ad libitum. The examination was done between 8.00 am to 3.00 pm. All experimental procedures were conducted according to the recommendations of the proper care and use of laboratory animals and following the recommendations of the National Institutes of Health guide for Care and Use of Laboratory Animals.

acute toxicity studies
Toxicity studies were carried out as per the OECD guidelines no 423 (8). No deaths or toxicity signs were reported after administering the extract orally in doses up to 2000mg/kg. Accordingly, different doses of 50, 100 & 200 mg/ kg body weight were chosen. Forty-two animals were erratically branched into two main groups (Unstressed and stressed groups) -Group I (Unstressed) a) Unstressed rats + saline (0.9% Nacl) b) Unstressed rats + Fluoxetine (1 mg/kg) -Group II (Chronic mild stress) (CMS) a) CMS rats + saline (0.9% Nacl) b) CMS rats + Fluoxetine (1 mg/kg) c) CMS rats +ethanolic extract of C.flexuosus (50 mg/kg) d) CMS rats + ethanolic extract of C.flexuosus (100 mg/kg) e) CMS rats + ethanolic extract of C.flexuosus (200 mg/kg) All drugs were given per os (p.o.) daily 30 minutes before exposing to daily schedule of CMS for 14 days (Table No.

1). induction of chronic mild stress (CMs)
Animals of the CMS group were exhibited to a routine of deep-rooted mild restlessness displayed beneath over a portion of time for 14 days period, in the period of time animals stayed in their hutch under usual circumstances. Behavioral activities were tested at the end of the experiment 10 .

Behavioral assessment of anxiolytic activity activity box
To judge the locomotor actions experiment was performed in a regular environment inside an activity box. Apparatus chosen for this work were created by clear Perspex (26x26x26 cm) embedded in powdered wood 11 . Trail was carried out in a noiseless area which was in the influence of white light as represented by 15 minutes earlier than monitoring the activity of animals were placed in the hutch to get adapted and reading were noted for 10 minutes.

Open field activity
The estimation of experimental actions in a new surrounding was carriedin an open field apparatus. Open field apparatus used in current experiment had a square area (76x76cm) with walls 42cm high. Ground was separated by bands into 25 equal squares 12 .
To evaluate the actions, rats were left in the center squarer of the open field. Numbers of square crossed with all four paws were noted for 5 minutes.

light dark box activity
Actions in a light-dark box were used as animal model of anxiety. The trial was performed in a locally made compartment box. The compartment of equal size (26 x 26 x 26 cm), with an access (12 x 12 cm) between the compartments, differed in their sensory properties. The coverings and walls of one compartment were light (transparent) and other dark (black) 13 . To examine the activity, rat was set in the center of the light compartment of the box. Entries and time consumed in the light compartment were observed for a close off time of 5 min. Entry into a compartment of the box is explained like the positioning of all four paws in the compartment of the activity box. elevated plus maze test Elevated plus maze was widely used to examine the tranquilizing actions.To carry out the plus maze model a specially designed apparatus was used. it was constructed in the lab. Totally, it consists of four arms in which two ere open and two were closed. Length (50cm) and breadth (10 cm) of the arms was kept identical and a connection was given from the center of5 cm 2 .Rat was held about 60 cm of peak from the ground in the center of the plus maze and time consumed and the entries in the open arm were computed for difference of 5 minutes 14 .

serotonin elisa test
At the end of the experiment, serum samples were collected for measuring serotonin levels using commercialized ELISA kits (cat. no. ab133053; Abcam) according to the manufacturer's protocol. The operational procedures were performed according to the manufacturer's instructions.

statistical analysis
Complete data was represented as mean ± SD and the figures were examined by using one-way analysis of variance (ANOVA) followed by Dunnett's "t" test for confirmation. P< 0.05 is considered as significance.

Phytochemical constituents in C. flexuous
Preliminary tests showed the presence of alkaloids, flavonoids, tannins, carbohydrates, glycosides and triterpenoids in C. flexuous as shown in Table No.2.

GC/Ms analysis
The ethanolic extract of dried leaves of C.flexuosus appeared as a dark green color liquid with very mild fragrant odor. The GC-MS analysis revealed the presence of esters (29.9%), acetals (15.05%), terpenes (10.49%), phytosterols (7.75%), aldehydes (7.25%) fatty acid hydrocarbons (5.97%), monosaccharides (4.68%), fatty acids (2.52%), fatty acid alcohols (2.54%) and phenolic compounds (1.36%). A total of forty-one compounds were identified in preliminary GCMS screening accounting for 90.58 % based on mass balance with respect to total peak area. Peaks below 900 points of match factor (SI) and reverse match factor (RSI) thresholds were not considered to compensate the internal standard corrections in Fig.1 Animals of the CMS group were exposed to a schedule of chronic mild stress shown above over a period of 14 days, while animals of unstressed group were kept in their cages. Group subjected to CMS model showed a significant decrease in locomotor and exploratory activity of the rats as compared to both unstressed animals given saline(192 crossing/10min) (*p<0.05) or fluoxetine(169.8 crossing/10min) ( a p<0.05) (1mg/kg). Stressed group given fluoxetine (1 mg/kg) showed (65.6) significant ( b p<0.05) increase in activity as compared to stressed groups given saline (9.6 crossing/10min). Stressed groups given C. flexuosus extract 50 mg/kg (88.4crossing/10min),100 mg/kg(122.8crossing/10min),200 mg/kg (95.8crossing/10min) showed a significant ( b p<0.05) increase in the locomotor activity when compared to stressed group (9.6crossing/10min). Group given C. flexuosus extract 100mg/kg (122.8crossing/10min) showed better effect than the two other doses and it also showed better effect than stressed group given fluoxetine. The results were depicted in Fig.2.

Open field activity
In theopen field test (OFT), Group subjected to CMS model showed a significant decrease in the number of square crossings frequencies(22 crossings/5min) as compared to both unstressed animals given saline (69crossings/5min) (*p<0.05) or unstressed animals given fluoxetine (34crossings/5min) ( a p<0.05). Stressed group given fluoxetine(1 mg/kg) showed significant ( b p<0.05) increase in number of square crossings(51 crossings/5min) as compared to stressed groups (21.8 crossings/5min). Stressed groups given C. flexuosus extract(50,100,200 mg/kg)showed a significant ( b p<0.05) increase in the number of square crossings (51, 59, 55 crossings/5min), respectively, ascompared to stressed group given saline (21.83 crossings/5min). Amongst the three doses of the test extract, 100 mg/kg was found to show better effect than the other two doses (50 mg/kg and 200 mg/kg) and the (CMS+Fluoxetine) group.The outcome of the anxiolytic effect was shown in Fig.3.

light and dark box
In the light and dark box, group subjected to CMS model without any treatment showed a significant decrease in time spent in light box of light dark transition box (9.6 sec.) as compared to both unstressed animals given saline (192 sec) (*p<0.05) or fluoxetine (169.8 sec) ( a p<0.05). Stressed group given fluoxetine (1 mg/kg) showed significant ( b p<0.05) increase in time spent in light box of light dark transition box(65.6 sec) as compared to stressed groups. Stressed groups given C. flexuosus extract 50,100,200 mg/kg showed a significant ( b p<0.05) increase in the time spent in light box of light dark transition box (88.4, 122.8, 95.8 sec) respectively, when compared to depressed group. Amongst the three doses of the test extract, 100 mg/kg was found to exert better effect as compared than the other two doses (50 mg/kg and 200 mg/kg body weight) and the (CMS+fluoxetine) group.The consequences of the anti-anxiety property was reflected in the Fig .4.

elevated plus maze
In the Elevated Plus Maze, group subjected to CMS model without any treatment showed a significant decrease in time spent in open arm of the elevated plus maze (11.6 sec) as compared to both unstressed animals given saline (192 sec) (*p<0.05) or fluoxetine (141.8 sec) ( a p<0.05). Stressed group given fluoxetine (1 mg/ kg) showed significant increase ( b p<0.05) in time spent in open arm of the elevated plus maze(85.6 sec) as compared to stressed group. Stressed groups given C. flexuosus extract 50,100,200 mg/kg Fig. 2. Activity cage test: Effect of C.flexuosus at a dose of 50, 100 and 200 mg/kg in stressed rats compared to fluoxetine 1mg/kg. All data represented as mean ± SD. * p < 0.05 compared to unstressed+saline group, a p < 0.05 compared to unstressed + fluoxetine group, b p < 0.05 stressed group. (n = 6)

disCussion
The current study aimed to investigate that whether administration of C.flexuosus ethanolic extract at three dose levels (50, 100 and 200 mg/kg) could ameliorate the behavioral deficits induced by chronic mild stress (CMS) in adult female wistar rats. The behavioral impairment was induced by CMS as it is considered a valid method of depression associated with anxiety 15 . The potential antidepressant and anxiolytic effect of C.flexuosus was estimated in refer to the standard SSRI; fluoxetine. Animals of the CMS group exposed to a schedule of chronic mild stress shown above over a period of 14 days showed a significant change in behavioral parameters, such as significant decrease inlocomotor activity and explorative activity, significant decrease in the number of  All data represented as mean ± SD. * p < 0.05 compared to unstressed+saline group, a p < 0.05 compared to unstressed + fluoxetine group, b p < 0.05 stressed group. (n = 6). square crossings frequencies, significant decrease in time spent in light box of light dark transition box and a significant decrease in time spent in open arm of the elevated plus maze. All out results were similar to previous studies conducted by Gihan et al,(2020) 16 . These behavioural deficit due to CMS model could be attributed to depression developed due to significant decrease in serotonin level as compared to control unstressed group 17 .
The current study showed that fluoxetine (1mg/kg)significantly increased the number of square crossings frequencies, time spent in light box of light dark transition box and time spent in open arm of the elevated plus maze in CMS animals. Same results was obtained in previous study 3 . It was also obvious that fluoxetine increased the locomotor activity in normal unstressed animal more than stressed animals and this result was obtained in a previous study and attributed to the limited beneficial effects or even adverse effects on anxiety and depression of acute fluoxetine administration 18 . These results are due to the serotonergic effect of fluoxetine that acts as an indirect agonist, stimulating multiple 5-HT receptors.
Stressed groups administered C. flexuosus at doses of (50, 100 and 200 mg/kg) showed a prominent antidepressant and anxiolytic effect with no significant difference than fluoxetine except for the extract at a dose of (100 mg/kg) that exerted better effect than fluoxetine and extract (50 and 200 mg/kg). The ameliorative effects of the extract were represented as significant increase in locomotor activity, number of square crossings frequencies, time spent in light box of light dark transition box and time spent in open arm of the elevated plus maze in CMS animals. As per the physiochemical study we can correlate these effects to the flavonoid content of the extract and to possible agonistic effect on GABA/benzodiazepine receptor complex, or via antagonizing the 5-HT1B receptor or agonize the 5-HT1A receptor 19 that was confirmed by the significant increase of serotonin level. In addition, we attributed the anxiolytic activity exerted by C. flexuosus to its contents of Geraniol; an acyclic monoterpene and the main component in the lemon grass oil, which was present in comparatively lesser levels in the leaf extract. It has been reported previously that geraniol showed antidepressant and anxiolytic activities 20,21 . The major constituent in the leaves extract, isoamyl acetate, and isovaleraldehyde diethyl acetal, finds their use as a flavoring agent and in perfume industry. P-hydroxybenzaldehye is known to have dopamine beta-monooxygenase inhibitor activity, which was found to abolish cocaine induced anxiety in animal models 22 . The nature and contents of chemical components in the leaf extract differed considerably form the literature 23,24 . Most of the GCMS analysis work available in the literature focuses on the study of extracted volatile oil from the leaves. It is imaginable that the method of anxiolytic action of C. flexuosus could be interceded by synergistic action of these phyto constituents.

ConClusion
It was concluded that ethanolic extract of C. flexuosus exerted a significant antidepressant and anxiolytic activities against stress induced in rats using CMS model. It was also reported repeatedly that administering the extract at a dose of 100 mg/kg orally gave the best effect amongst the three doses as well as better effect when compared to fluoxetine, which is an accredited standard for treatment of anxiety. Additional molecular studies can validate the precise underlying mechanism.
We also recommend further clinical studies for the management of stress and muscle tension problems.

aCKnoWledGeMent
The authors gratefully acknowledge Dr. Sailaja Rao, Assistant Professor, College of Pharmacy, Jazan University for her support in reviewing this article. Complete preliminary and pharmacological work was carried out in the college of pharmacy, Jazan University. The authors are thankful to the authorities of Future scientist 6 -Jazan University -KSA for funding the project to carry out the successful trails.

Conflict of interest
Authors declared that there is no conflict of interest Contribution of authors I/we declare that this work was done by the author(s) named in the article and all the liabilities pertaining to claims relating to the content of this article will be borne by the authors.