Dose-Dependent, Antidepressant, and Anxiolytic Effects of a Traditional Medicinal Plant for the Management of Behavioral Dysfunctions in Animal Models.

The present work was carried out to assess the Onosma bracteatum anxiolytic and antidepressant properties. Swiss albino mice (male) were fed orally with hydroalcoholic extract at different doses 50, 100, and 200 mg 1 hour prior to test with the standard diazepam and fluoxetine. Anxiolytic and antidepressant activities were evaluated by using open field, elevated plus maze, force swimming, and tail suspension test. Results of open field test showed an increase in number of line crossing as well as number of rearing in dosage-dependent design. Although results of elevated plus maze test evidently showed antianxiety effect of O bracteatum by increasing the time spent in open arms along with decreasing the time spent in closed arms in dosage-dependent way. For the evaluation of antidepressant effect, O bracteatum diminished the immobility time and expanded mobility time in forced swim model in dosage-dependent way. Likewise, O bracteatum expanded time span of mobility along with diminished immobility time in tail suspension method in dosage-dependent way. Outcome demonstrated that plant at the dose of 200 mg/kg body weight showed significant potential which was similar to that standard diazepam and fluoxetine. Hence, O bracteatum may be used as potent natural psychotherapeutic agent against the mental disorders.

Introduction

Depression and anxiety are the two most common mental disorders.[1-3] Anxiety as well as depression presents the most widely recognized types of mental sicknesses nowadays, influencing very nearly 10% to 20% of the total population.[4] Some currently used psychological therapies effective in treating depression and anxiety includes.

Serotonin reuptake inhibitors, for example, paroxetine, serotonin–norepinephrine reuptake inhibitors, for example, venlafexine, monoamine oxidase inhibitors, for example, phenelzine and tricyclic and tetracyclic antidepressants; for example, amitriptyline benzodiazepines.[5,6]

Regardless of the accessibility of various classes of medications for the treatment of depression and anxiety, full reduction in clinical feature has stayed subtle. Clinical utilization of these medications is restricted by their adverse effects and underprivileged tolerability profile. The viability, duration effect, and adverse effects of the existing medications have established genuine concern and the requirement for more up-to-date medications. The decent variety in neural targets makes phytomedicine a promising possibility for the management of these sicknesses. These days, there appears to be overdependence on modern medications for restoring or improving definite mental disorders. In the meantime, researches have demonstrated that numerous individuals hoping for herbal preparations for the treatment of various types of mental issues. Recently, scientific studies are being centered on the approval of popularly acclaimed herbs with psychoactive properties.[7]

Due to less adverse effects and more friendly to human body, the medicinal plant–based preparations are utilized for basic health care in most of the underdeveloped countries. Almost 80.0% of the populace utilizes the herbs for this purpose.[8] In Pakistan, Unani medicine has been used for the treatment of various ailments. Unani medicine possesses a lot importance for its use in health care throughout the country either tribal or metropolitan.[9]

Onosma bracteatum wall, has a spot with the family Boraginaceae usually fampus as “Gaozaban” or “Sedge,” found in Asian nations including Pakistan and well-known for its effects on heart and mental problems.[10] It develops in dry or sodden and sunny climate for the most part in rock crevices and generally called as rock garden herb.[11] Onosma bracteatum is accounted for to have important active compounds such as flavonoids, tannins, sugars, phenolic, and glycosides. It is consumed as main ingredients in various Unani as well as Ayurvedic preparations used in the treatment of various disorders with respect to human health.[12,13] This medicinal plant is greatly utilized in the management of anxiety and other psychological disturbances, immune system, and hormone-related disturbances that lead to several chronic ailments such as asthma, hypertension, Parkinsonism, diabetes, cardiac disorder, and cancer.[14-17]

The aim of the current research was to screen the native herbs, especially used as traditionally for their antianxiety and antidepressant potential, as Pakistan is enriched with diverse flora of plants. Onosma bracteatum was chosen based on their historical utilization as calming and aphrodisiac agent. Aforementioned herb, if explored scientifically, might be a wellspring of modest and compelling to treat the previously described issue which may likewise open latest skyline in the field of management.

Methods

Plant Material and Preparation of Crude Extract

Plant was bought from Shahi Bazaar of Bahawalpur and identified by Prof Dr Ghazala H. Rizwani, Director Research, Hamdard University, Karachi, Pakistan. After cleaning of the plant material, plant was dried under shade. Example tests of dried plant material were stored in the Herbarium of Pharmacognosy investigate research facility, Faculty of Eastern Medicine, the Hamdard University Karachi, Pakistan, and reference number for the plant (A141) was issued for future reference. Squashed material of plant was dipped in watery ethanol solvent (30:70) at room temperature, with intermittent stirring. Dipped plant material was then separated through muslin fabric first and Whatman #1 filter paper. The procedure toward drenching along with filtration was done again at 2 additional occasions. The remainder of plant material was then disposed of and gathered filtrate was focused by dissipating first under rotating evaporator and a short time later in an oven, thick paste-like semisolid form of extract was obtained. Crude extract of plant was then named, evaluated to acquire percentage yield, and put away in cooler for sometime later use.

Drugs

Aqueous ethanolic extract of O bracteatum at dosages of 50, 100, and 200 mg/kg were given individually 1 hour before the activity. Diazepam (DZP; 1 mg/kg, Sigma, St. Louis, USA) was used as standard anxiolytic drug. Fluoxetine (10 mg/kg, Sigma) was used as standard antidepressant agent. Normal saline (10 mL/kg) was used for the treatment of the control groups.

Experimental Animals

Swiss albino mice (male) having their weights between 22 and 42 gm were utilized in the research and purchased from the animal house of the Faculty of Pharmacy and Alternative Medicine. All the animals were kept in the animal house experimental area of pharmacology research laboratory of Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur. Standard research center conditions (12-hour light/dim cycles at 22 ± 2°C) were kept up for creatures and they were nourished with standard food and water ad libitum. Rules affirmed by Institutional Animal Ethics Committee of the Islamia University Bahawalpur were pursued for conduction of research.

The study was approved by the ethical committee of University College of Conventional Medicine, Faculty of Pharmacy and Alternative Medicine, The Islamia University of Bahawalpur. Wide notification number 7117/Pharm dated October 24, 2018.

Experimental Groups

All the animals were divided into 5 different treatment groups consisting of 5 animals in each group. Group I named as control and just given vehicle NaCl 0.9% normal saline, group II called as standard and received DZP (1 mg/Kg) in antianxiety activity, and Fluoxetine (10 mg/kg) in antidepressant activity. Group III to V is of different dosages of the plant extract (50, 100, and 200 mg). Every one of the medications was administered by oral route 1 hour before the test.

Evaluation of Anxiolytic Activity of O bracteatum

Open field test

The open field test (OFT) model was developed of white compressed wood and estimated 32 cm × 32 cm with 18 cm dividers. One wall was plain Plexiglas, so animals might be noticeable in the model. Blue colored lines were drawn on the bottom with the help of a marker as well as were noticeable from first to last of the apparent Plexiglas floor. The lines partitioned the bottom into sixteen 12 cm × 12 cm squares. A focal square (12 cm × 12 cm) was attracted at the center of the open field. The focal square is utilized on the grounds that some mice strains have good locomotor action and cross the lines of the test compartment again and again throughout a test session. Likewise, the focal square has adequate space encompassing it to offer importance to the focal area as being distinctive commencing the external areas. The maze was situated in a 1.8 m × 4.6 m test room and lit by a 60-watt red light for foundation lighting. The open field maze was cleaned between the each mice utilizing 70% ethyl alcohol. The open field was utilized to assess the exploratory movement of the creatures. The watched parameters were the quantity of lines crossed (with 4 paws) and number of rearings.[18]

Elevated plus maze test

Elevated plus maze test (EPMT) is most broadly utilized as well as approved method to quantify anxiety in animal models. Mechanical assembly comprised of 4 arms of which 2 were remained open along with 2 shut. Open arms (35 cm2 × 5 cm2) were crossed with shut arms (35 cm3 × 5 cm3 × 20 cm3) at an inside point (5 cm2 × 5 cm2). Elevated plus maze was raised to a tallness of 50 cm starting from the earliest stage set in a room having faint brightening. Creatures were treated with separate treatment groups and following half-hour, they were exclusively put on EPM device at the middle, confronting one of the shut arms. Duration (in a moment or 2) spent by every one of the animal on open and shut arms was noted for 300 seconds.[19,20]

Evaluation of Antidepressant Activity of O bracteatum

Forced swim test

Forced swim test (FST) is the most generally utilized as well as approved in vivo method for estimation of antidepressant effect. Device comprised of straight forward Plexiglass chamber (20 cm × 12 cm). It was loaded up with water (24°C ± 1°C) to 15 cm depth.[21]

A pretest swimming session was given to all the animals for 15 minutes every 1 day prior to definite test session. Animals were treated with particular gathering medicines following pretest session, 6 hours prior to definite investigation session, and half-hour prior to conclusive investigation session. Half-hour after definite portion or one day after pretest session, every creature was independently put in Plexiglass chamber for conclusive swimming session of 5 minutes each. Term of fixed status was observed for every creature for 300 seconds. Animals were viewed as fixed when no endeavors were made to get away, except developments important to stay its head out of water.[22]

Tail suspension test

Tail suspension test (TST) mechanical assembly comprised of wooden chamber (70-cm high). A bar was fitted between side dividers of load, at a tallness of 60 cm from ground or 10 cm from top of the mechanical assembly. Creatures were hung with the pole by putting sticky tape 1-in from tip of tail. Creatures were given a pretest session of 15 minutes every 1 day before definite test session. Creatures were treated with separate gathering medicines following pretest session, 360 minutes before conclusive test session and half-hour before definite test session. Half-hour after definite portion or 1 day after pretest session, every creature was separately hung with pole for conclusive test session of 300 seconds each. Term of stability was noted for every creature for 300 seconds. Mouse was viewed as stable when it inactively hung with bar without any endeavors to get away.[23-26]

Acute Toxicity Study of O bracteatum

Rules of Organization for Economic Cooperation and Development were pursued to execute acute lethality studies.[27] Mice (22-42 g) were partitioned into 5 different groups, comprising of 5 mice in each group. Ordinary behavioral parameters of all the animals were noted. Investigational creatures were fasted during night but got water ad libitum. One group of mice got normal saline (10 mL/kg) and further 4 groups got O bracteatum plant extract at the dosages of 1, 3, 5, and 10 g/kg individually. Behavioral changes along with reaction parameters; for example, hyperactivity, alertness, convulsions, grooming, lacrimation, sweating, pee, corneal reflex, righting reflex, pain response, contact reaction, gripping quality, and mortality were watched and recorded at 30 minute, 1, 2, 4, 6, 12, 24, and 48 hours.[28,29]

Statistical Analysis

Data were exhibited as mean ± standard error of the mean. The analysis of the outcomes was completed by exposing the information to measurable investigation utilizing one-way analysis of variance. All of the outcomes were analyzed utilizing GraphPad Prism programming version 5. Impacts were viewed as huge where P < .05.

Results

Results of Anxiolytic Activity

Results of anxiolytic activity of O bracteatum using OFT

The OFT was selected to investigate the action of the plant extract upon spontaneous motor activity. Overall number of line crossings along with rearings into OFT by animals of the standard as well as treatment groups were judged against with that of control group in 5-minute activity. Observation was done after 1 hour of the drug administration and results were noted. Onosma bracteatum exhibited dose-dependent enhancement in number of line crossings along with number of rearings (Table 1).

Table 1.

The Effects of the Crude Extracts of Onosma bracteatum on Number of Line Crossings and Rearings on Open Field Test and on Time Spent in Open Arms and Time Spent in Closed Arms in Elevated Plus Maze Model.a

Group	Open Field Test		Elevated Plus Maze Model
	Number of Line Crossing	Number of Rearing	Time Spent in Open Arms (s)	Time Spent in Closed Arms (s)
Control (N/S 10 mL/kg)	87.16 ± 7.0 (NS)	34.12 ± 13 (NS)	32.14 ± 3.05 (NS)	262.19 ± 3.11 (NS)
Standard (DZP 1 mg/kg)	133.83 ± 9.10b	59.02 ± 10b	104 ± 8.14b	191.5 ± 8.99b
Onosma bracteatum (50 mg/kg)	87.1 ± 3.2 (NS)	34.12 ± 8.0 (NS)	50.49 ± 5 (NS)	243.81 ± 5.1 (NS)
Onosma bracteatum (100 mg/kg)	98.99 ± 6.96 (NS)	42 ± 5.2 (NS)	74.5 ± 5.43c	220 ± 5.79c
Onosma bracteatum (200 mg/kg)	116.90 ± 7.8c	49 ± 6.5c	91.5 ± 6.41d	203.5 ± 6.24d

Abbreviations: ANOVA, analysis of variance; DZP, diazepam; NS, normal saline; SEM, standard error of the mean.

a The data are expressed as mean ± SEM of 6 creatures in each gathering. The outcomes are examined utilizing 1-way ANOVA and contrasted with those of normal control gathering.

b The outcomes are viewed as exceptionally significant, if P < .001.

c The outcomes are viewed as noteworthy, if P < .05.

d The outcomes are viewed as increasingly significant, if P < .01.

Results of OFT undoubtedly show the anxiolytic action of O bracteatum. Onosma bracteatum increases the number of line crossing as well as number of rearing in dosage-dependent design. Results showed that O bracteatum plant extract at 200 mg/kg dosage possessed comparable effect with the diazepam.

Results of anxiolytic effect of O bracteatum utilizing elevated plus maze model

Time spent in open and closed arms by animals of standard as well as treatment groups, in 5-minute duration, was contrasted with that of normal control group. Observation was done after 1 hour of the treatment and results were noted.

Antidepressant Activity of the Crude Extracts of O bracteatum

Results of antidepressant activity of O bracteatum using forced swim model

Time span of mobility along with immobility shown by animals of standard as well as treatment groups, in 300-second swimming session, was contrasted with that of normal control group. Onosma bracteatum diminished the immobility time and expanded the mobility time in dosage-dependent way (Table 2).

Table 2.

The Effects of the Crude Extracts of Onosma Bracteatum on Duration(s) of Mobility and Immobility in Forced Swim Model and in Tail Suspension Model.a

Group	Forced Swim Model		Tail Suspension Model
	Duration of Mobility(s)	Duration of Immobility(s)	Duration of Mobility(s)	Duration of Immobility(s)
Control (N/S 10 mL/kg)	138.4 ± 8.6 (NS)	161.6 ± 8.6 (NS)	66.50 ± 7.14 (NS)	233.5 ± 7.14 (NS)
Fluoxetine (FXT 1 mg/kg)	247.1 ± 5.90b	52.9 ± 5.90b	223.4 ± 3.45b	76.6 ± 3.45b
Onosma bracteatum (50 mg/kg)	167.4 ± 4.32c	132.6 ± 4.32c	120.9 ± 5.20 (NS)	179.1 ± 5.20 (NS)
Onosma bracteatum (100 mg/kg)	195.5 ± 4.79d	104.5 ± 4.79d	141.8 ± 6.14c	158.2 ± 6.14c
Onosma bracteatum (200 mg/kg)	224.1 ± 4.49b	75.9 ± 4.49b	167.1 ± 7.01d	132.9 ± 7.01d

Abbreviations: ANOVA, analysis of variance; NS, normal saline; SEM, standard error of the mean.

a The values are communicated as mean ± SEM of 6 creatures in each gathering. The outcomes are examined utilizing 1-way ANOVA and contrasted with those of normal control gathering.

b The outcomes are viewed as exceptionally significant, if P < .001.

c The outcomes are viewed as noteworthy, if P < .05.

d The outcomes are viewed as increasingly significant, if P < .01.

Result of antidepressant activity of O bracteatum using tail suspension model

Time frame of mobility as well as immobility in animals of standard along with the treatment groups was contrasted and that of normal control group. Onosma bracteatum likewise expanded the time span of mobility and diminished the immobility in dosage-dependent way (Table 2).

Acute Lethality Study of O bracteatum

The crude concentrate of O bracteatum was given intraperitoneally to the animals at dosages of 1, 3, 5, along with 10 g/kg. No indications of poisonous quality and mortality were seen up to 10 g/kg dosage.

Discussion

Active compounds found in various pieces of plants have been appeared to have remedial qualities. Utilizing entire plant as a medication would thus be able to be utilized to receive greatest helpful rewards.[30] Flavonoids found in various medicinal plants have been appeared to have antianxiety and depression reducing impact, for example, Hypericum perforatum.[31]

In the current research, hydroalcoholic extract of O bracteatum prepared from the leaves demonstrated anxiolytic effect in OFT and in EPMT. In OFT, O bracteatum increases the number of line crossing as well as the number of rearings in dose-dependent design. Results of 200 mg/kg dosage of plant extract O bracteatum were comparable with the diazepam. While in EPMT, O bracteatum expanded the time spent in open arms as well as diminished the time spent in shut arms in dosage-dependent way. Anxiolytic capability of O bracteatum 200 mg/kg was practically significant to that of diazepam as indicated by elevated plus maze model.

In present investigation, forced swim model and tail suspension models (TSMs) were utilized in Swiss Albino mice for assessment of anxiolytic action of plant extract of O bracteatum. Results reported that 200 mg/kg dosage of O bracteatum indicated potent antidepressant effect (P < .005) that was similar to that of fluoxetine. The outcomes of forced swim as well as TSM affirm considerable antidepressant capability of O bracteatum. Promising mechanisms for antidepressant effect of crude extracts of the plant accounted may be due to inhibitory action on monoamine oxidase A. Fluoxetine showed marked antidepressant potential due to selective inhibition of serotonin reuptake.

Fajemiroye et al announced that the Nymphaea lotus treated mice invested factually more duration in the open/exposed territories of the maze and augmented EPM arm entry which were measurably significant when contrasted with the control. Fajemiroye et al results are comparable with the consequences of O bracteatum and demonstrate possible anxiolytic impacts by O bracteatum.[32]

A study was conducted by Rajput and Khan also announced that, Nelumbo nucifera fruit extract in EPMT caused a considerable increment in number of open arm crossings and duration spent in open arms at 100 and 200 mg/kg which was profoundly noteworthy. Onosma bracteatum leaves extract additionally showed significant comparative outcomes when contrasted and the N nucifera fruit extract at 200 mg/kg. Rajput and Khan exhibited that N nucifera fruit extract demonstrated a noteworthy decrease in the time span of immobility of mice at dosages 100 and 200 mg/kg in FST. The consequences of the present research are practically comparable at 200 mg/kg dose with that of the Rajput and Khan results.[22]

Already, Diniz et al reported that essential oil of Annona vepretorum in EPMT demonstrated the significant rise in the quantity of entries in the open arm at 100 mg/kg, while OFT demonstrated a noteworthy decrease in the quantity of line crossing at 100 mg/kg when contrasted with the standard DZP. Moreover TST showed a significant reduction in immobility time when comparison was done with the standard. Consequently, the results of our investigation are up to the degree of Diniz et al results which showed the significant anxiolytic and antidepressant effect of the O bracteatum.[33]

Maggu et al likewise announced that ethanol extract of Juglans regia indicated significant dose-dependent anxiolytic and antidepressant impact at 200 mg/kg, and chloroform extract of Prunus amygdalus simply revealed anxiolytic action at 200 mg/kg dose. Onosma bracteatum likewise indicated significant anxiolytic and antidepressant potential and is similar to the Maggu et al result.[34]

From present research, it is concluded that O bracteatum showed significant anxiolytic and antidepressant effect in dose-dependent way. Therefore, the present study validates the historic use of the O bracteatum as an anxiolytic and antidepressant agent that were used in the traditional system of medicine. Further studies are needed to evaluate the chemical constituents that are responsible for the activity and to know its mechanism of action.