Bioactive metabolites of Blumea lacera attenuate anxiety and depression in rodents and computer‐aided model

Abstract Blumea lacera is an edible plant with imperative medicinal values. However, the anxiolytic and antidepressant roles of B. lacera have not been well‐explained. Therefore, the current study aims to explore the impending bioactive metabolites and roles of B. lacera methanol leaf extract (Me‐BLL) in attenuating anxiety and depression through several experimental and computer‐aided approaches. The chemical characterization of Me‐BLL was performed through standard phytochemical and GC‐MS analyses. To explore the neuropharmacological insights, Swiss albino mice were treated with Me‐BLL at doses of 200–400 mg/kg, p.o. The anxiolytic effects were observed employing elevated plus maze (EPM), light–dark box (LDB), and hole‐board (HBT) tests, while antidepressant effects were evaluated using forced swimming (FST) and tail suspension tests (TST). Diazepam (1 mg/kg, i.p.) and fluoxetine HCl (20 mg/kg, p.o.) were used as the reference standard. The phytochemical analyses revealed several bioactive metabolites, including higher contents of total phenolics and flavonoids. The EPM and LDB tests demonstrated an increased time spent in open arms and light box, and the HBT showed an increased number of head dipping, indicating the anxiolytic effects of Me‐BLL. The TST and FST revealed a decrease in immobility time, meaning the persuasive antidepressant effects. The antioxidative effects of Me‐BLL have also been observed prominently. Correspondingly, the computer‐aided investigation confirmed several bioactive lead molecules. Specifically, thymol and cuminol revealed potential anxiolytic and antioxidant effects, while stigmast‐5‐en‐3.beta.‐ol and gamma‐sitosterol possessed promising antidepressant effects. Taken these results as a base, the plant has imperative potentials in managing anxiety and depression‐like disorders.


| INTRODUC TI ON
More than 450 million people suffer from psychiatric or behavioral disorders, which add up to 12.3 percent of the global disease burden and are expected to increase to 15 percent by 2020 (Rajput et al., 2019). Currently, the most psychiatric conditions that raise morbidity in the world tend to be anxiety and depression. In addition, the WHO has rated depressive disorders as the world's leading source of non-fatal health conditions, with anxiety disorders ranking sixth (WHO, 2017). Multiple studies indicated that both anxiety and depression happen simultaneously and do not represent distinct disease entities. Around one portion of those researched with depression are also diagnosed with anxiety disarray.
The leading causes of anxiety and depression remain a great conundrum. Still, few prevalence factors like genetic, environmental, biological, and psychological have been unfolded to be connected in the progression of such neuropsychiatric disorders (Berton & Nestler, 2006). Various pathophysiological events occur as a result of changes in the γaminobutyric acid (GABA)ergic, serotoninergic, and glutamatergic systems. The GABA acts as the vital modulator mechanism in the central nervous system (CNS), an impact that is balanced by glutamate (with an excitatory activity). A down-regulation of GABAergic transmission has been linked to anxiety disorders; thus, many anxiolytic drugs have this system as a target of action.
Its activity results from the recognition of two types of receptors: the ionotropic GABA-A and metabotropic GABA-B receptors. Many physicians prescribed benzodiazepines as anxiolytics that act on the GABA-A, but these generate many adverse effects such as sedation, motor incoordination, cognitive impairments, tolerance, and addiction (Barker et al., 2004). Besides, the 5-hydroxytryptamine (5-HT) is a key modulatory neurotransmitter associated with the pathophysiology and treatment of anxiety (Resstel et al., 2009). Therefore, the antidepressants which modulate the 5-HT reuptake (SSRIs) are acted as anxiolytics. Notably, the 5-HT1A receptor is widely expressed, and it is associated with anxiety disorders.
However, present top-notch neuropsychiatric medications (e.g., benzodiazepines, selective serotonin, and/or serotoninnorepinephrine reuptake inhibitors) cannot offer adequate clinical procedures to simultaneously modulate anxiety, depression, chronic inflammation, and reactive oxygen species (ROS) (Penn & Tracy, 2012). In addition, these medications have adverse side effects, including sedation, sexual dysfunction, memory disruptions, amnesia, and daytime drowsiness, which was of considerable concern to society (Wang et al., 2018). Throughout the quest for new remedial products for the treatment of neurological disorders, herbal/ medicinal plant research has also led mainly to demonstrating the pharmacological efficacy of various herbs throughout different animal models (Zhang, 2004). In addition, the discovery of potentially bioactive compounds from herbal products has broad targets of pharmacological responses that are the focal point of recent global research interest (Lee & Kim, 2016). As developments of the drug are involved in the discovery of lead compounds from natural products, while it was followed by lead identification, lead optimization, lead development, finally it approaches for successfully consecutive clinical trials, and the compounds also approved for clinical application (Balunas & Kinghorn, 2005).
Blumea lacera (Burm.f.) DC., belonging to the Asteraceae family, has an enormous medicinal value, and the leaves have been widely used in the traditional medicinal system. The leaf is edible and the most used part of the herb. The juice of leaves is antispasmodic, anthelmintic, astringent, febrifuge, stimulant, and diuretic; and able to cure bronchitis, fevers, and burning sensation (Khare, 2008).
Nonetheless, so far, little work has been conducted to explore its pharmacological potential. Therefore, this research examined the antioxidant, anxiolytic, and antidepressant activity of B. lacera systematically alongside its chemical characterization and pursued computer-aided approaches to unravel the possible bioactive lead molecules of the plant.

| Animals
Five to six weeks old male Swiss albino mice weighing between 22 to 30 g were acquired from the animal research division of the International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR,B). Animals were housed in poly-carbonated cages, ensuring a standard laboratory condition (room temperature 23 ± 2℃) and humidity 55%-60% in a 12 hr/daylight cycle (Akter et al., 2021). They had free access to diet and tap water supplied with pellets. The mice had been acclimatized (14 days) to adapt to the laboratory environment before starting the experiments.

| Extract preparation
The collected leaves were cleaned and dried under shade ground at room temperature (23 ± 0.5℃). The dried samples ground to a coarse powder with a mechanical grinder, and then, around 500 g powder successively extracted with methanol to yield crude extract.
Finally, the filtrate was evaporated by a rotary evaporator (RE200, Bibby Sterling, UK) under reduced pressure and temperature below 50℃. The methanol extract (Me-BLL) yield value was noted as 16 g, and until the experiment was conducted, Me-BLL was stored at 4℃ (Hossen et al., 2021).

| Qualitative phytochemical screening
The qualitative phytochemical analysis has been performed following the standard protocol (Akter et al., 2021;Khan et al., 2020) to confirm the presence of various secondary metabolites in Me-BLL.
The flow rate of the column was 0.6 ml/min Helium gas at a constant pressure of 90 kPa. The aux (GC to MS interface) temperature was 280℃. The MS was set with a scanning mode with a scanning range of 40-350 amu, while the ionizing mode was in the form of electron ionization (EI) and the range of mass set within 50-550 m/z. One μL of the sample was injected in split fewer modes. Complete GC-MS running time was set for 29.33 min, and the compounds in the peak areas were identified by comparison to those in the GC-MS library version NIST 08-S database.

| Determination of total plant phenolics
The concentrations of phenolic compounds in Me-BLL were determined following the established method (Ali Reza et al., 2018;Esmaeilzadeh Kenari et al., 2014). The 0.5 ml of plant extract or standard solution at different concentrations was added to 2.5 ml of Folin-Ciocalteu (diluted ten times with water) reagent and 2.5 ml of Na 2 CO 3 (7.5%) solution. The reaction mixture was incubated for 20 min at 25℃, and the absorbance of the mixture was measured at 760 nm. The experiment was performed in triplicate, and findings were stated as mean ± SEM, and values are expressed as mg of gallic acid equivalent (GAE)/g of dried extract.

| Determination of total plant flavonoids
The total flavonoid contents of the Me-BLL were investigated by the aluminum chloride colorimetric method described previously (Ali Reza et al., 2018;Rajaei et al., 2021). One mL from each concentration of the plant extract was added to 3.0 ml of methanol, 0.2 ml of 10% AlCl 3 , 0.2 ml of 1 M potassium acetate, and 5.6 ml of distilled water. The reaction mixture was then incubated at room temperature for 30 min to complete the reaction. The absorbance of the mixture was measured at 420 nm. The study was conducted in triplicate, and results were reported as mean ± SEM, and values are expressed as mg of quercetin equivalent per gram (QE/g) of dried extract.

| Evaluation of antioxidative potential
2.7.1 | DPPH free radical scavenging assay The free radical scavenging activity of Me-BLL was determined by DPPH assay according to the method described previously Rashid Chowdhury et al., 2021). Two mL of methanol solution of plant extract or reference standard ascorbic acid at different concentrations was mixed with 3 ml of methanol solution of DPPH (4 mg in 100 ml methanol) into the test tube. The reaction mixture was incubated at room temperature for 30 min in a dark place to complete the reaction. The absorbance of the solution was measured spectrophotometrically at 517 nm. DPPH free radical scavenging ability (%) was calculated by using the formula: 2.7.2 | Hydroxyl radical scavenging assay The hydroxyl radical scavenging action of Me-BLL was determined by the method as described earlier (Ali Reza et al., 2018;Haida & Hakiman, 2019). A 3 ml of the reaction mixture was prepared with 1 ml of 1.5 mM FeSO 4 , 0.7 ml of 6 mM H 2 O 2 , and 0.3 ml of 20 mM sodium salicylate to dilute at 50-800 μg/mL. The mixture was read at 562 nm after one h incubation at 37℃ against an appropriate blank solution. Hydroxyl radical scavenging ability (%) was calculated by using the formula: (absorbance of control − absorbance of sample) ∕absorbance of control × 100.
(absorbance of control − absorbance of sample) ∕absorbance of control × 100 2.7.3 | Assay of iron-chelating effect The iron-chelating effect of Me-BLL was evaluated compared with ascorbic acid, and also the whole test was administrated according to the established procedure (Akhter et al., 2015). Briefly, both the test sample and ascorbic acid (50-800 μg/mL) were added to Ophenanthroline (0.05%) and ferric chloride (200 μM) solution while the crude samples excluded in control. The reaction mixture was read at 510 nm after 10 min incubation at room temperature. The following equation calculated the percentage of iron-chelating activity of Me-BLL:

| Acute oral toxicity test
The acute oral toxicity test was performed following the OECD guidelines and established protocol. The allocated animals (n = 5) were administered a single oral dose (100 to 2000 mg/kg, body weight) of the test extract (Me-BLL). Prior to administering the extract, mice were kept fasting overnight, and food was also delayed between 3 and 4 hr. After administration, food was withheld for a further 3-4 hr. Experimental animals were observed individually during the first 30 min after dosing, periodically for the first 24 min (special attention for the first 4 hr), with special monitoring for possible unusual responses including behavioral changes, allergic syndromes (itching, swelling, skin, and rash), and mortality over the next 72 hr. The median therapeutic effective dose was intervened as one-tenth of the median lethal dose (LD50 >2.0 g/kg) (Al-Araby et al., 2020).

| Experimental design
For each experiment, a total of twenty mice were separated into four groups (Group-I to IV) containing five mice (n = 5) in each group.

| Light and dark box test
The light and dark box test (LDB) test helps in predicting whether the anxiolytic or anxiogenic properties are present in laboratory animals.
The test was performed following the established method described earlier ). An animal activity monitor outfitted with two-compartment test chambers housed in a dark, air-conditioned room was used to conduct the test. The light-dark box is designed with an open-topped rectangular box consisting of a floor area of 46 × 27 ×30 cm 3 that is divided into two parts, small (19 × 27 cm 2 ) and a large (27 × 27 cm 2 ) area. An 8 × 8 cm passageway provided access between the two compartments. Each light compartment was illuminated with a 60W red tungsten bulb at the height of 30 cm above the test chamber's door. The time spent in the compartments was monitored and recorded. Animals (n = 5) of each group were administrated as per the experimental design. After 30 min treatment with control, diazepam, and treatment group, each mouse was set onto individually in the test chambers, and their activity was recorded over 5 min.

| Hole-board test
The method applied for hole-board test (HBT) was similar to those described earlier Rashid Chowdhury et al., 2021).
A grid pattern with sixteen holes (diameter 3 cm) in this model included a flat platform with an enclosed area (20 x 40 cm 2 ) used as an experimental apparatus set up 15 cm above the floor. Dosing treatments have been followed for each group of animals according to the experimental design indicated. The experimental animals were placed in the center of the board thirty minutes after the test dose was administered and permitted free movement. Finally, the head dipping through the holes and the latency of mice dipping the head was counted for 5 min.

| Forced swimming test (FST)
According to this method, mice were forced to swim independently in an open glass compartment (10 × 15 cm 2 , d × h) containing fresh water with a depth of 19 cm and kept at (25 ± 1) °C. Mice of all groups were treated as per the statement of the experimental design section. After thirty minutes, each mouse was placed in the tank for 6 min, where the first 2 min was considered initial adjustment time, and the next 4 min was recorded as the immobility duration .

Docking tools
The molecular docking was performed using Schrodinger suites-Maestro 2017-1. The Pockdrug online server was used to predict the best binding pocket and probable drug ability. Discovery studio (v 4.1) was used for the visualization.

Ligand preparation
The chemical structures of eight major compounds were extracted from the PubChem repository (https:/pubchem.ncbi.nlm.nih.gov/).
The ligand was prepared using the LigPrep tool, embedded in Schrödinger suite-Maestro v 11.1, where the following parameters were used for minimization: neutralized at pH 7.0 ± 2.0 using Epik 2.2 and the force field OPLS3.

Receptor/Enzyme preparation
Three-dimensional crystallographic structures of enzyme/receptors were obtained from the Protein Data Bank RCSB PDB: urate oxidase (Uox) enzyme receptor (PDB: 1R4U), potassium channel receptor (PDB: 4UUJ), and human serotonin receptor (PDB: 5I6X). Preprocessing, optimization, and minimization processes were done by using Protein Preparation Wizard. This process is included in the Schrodinger suitmaestro (v11.1). The structures were optimized at pH 7.0, and water molecules fewer than 3 H-bonds to non-waters were removed.
Restrained minimization was done where heavy atoms are converged to an RMSD of 0.30 Å on the implemented OPLS3 force field. Then the receptor grids were generated after selecting the best binding sites by using an online tool, PockDrug (Hussein et al., 2015).

Glide ligand molecular docking
The molecular docking was performed to select the better ligand that can further be studied comparing with the standard drugs for determining the antioxidant, anxiolytic, and antidepressant effects.
The docking was done by ligand docking option using Schrodinger suite-maestro (v11.1) .

| Evaluation of pharmacokinetic parameters
The absorption, distribution, metabolism, excretion, and toxicity (ADME/T) properties analysis of Me-BLL were evaluated by the Lipinski's rule of fives and Veber's rules (number of rotatable bonds; topological polar surface area). In addition, the ADME/T properties analysis was evaluated by SwissADME (http://www.swiss adme.ch/) (Sakib et al., 2021).

| Determination of toxicological properties
AdmetSAR online tool was used to determine the toxicological properties of the selected compounds, while a prime concern during the development of new drugs is toxicity (Yang et al., 2019). In this study,

| Statistical analysis
The data were analyzed by one-way analysis of variance (

| Qualitative phytochemical screening
The qualitative phytochemical screening of Me-BLL unveiled the presence of several secondary metabolites, including alkaloids, carbohydrates, flavonoids, phenols, protein, and amino acids, diterpenes in Me-BLL (Table 1).

| GC-MS analysis
The GC-MS analysis of Me-BLL revealed around thirty secondary metabolites having retention time between 9.16 and 29.09 as listed in Table 2

| Total plant phenolics and flavonoids
Quantitative analyses revealed that the Me-BLL contained the highest phenolic content (32.27 ± 1.06 mg of GAE/g of dried extract). At the same time, Me-BLL also has the highest flavonoid content (172.35 ± 0.25 mg of QE/g of dried extract) (Table 3).

| DPPH free radical scavenging assay
The study explored significant scavenged DPPH radicals in a dosedependent manner (Figure 1a). Me-BLL showed the most potential scavenging activity against DPPH radicals with an IC 50 value of 133.48 ± 3.67 μg/mL, which is comparable to that of the reference standard ascorbic acid (AA) having an IC 50 value of 103.16 ± 0.56 μg/ mL (Figure 1a).

| Hydroxyl radical scavenging assay
The hydroxyl radical scavenging activity of the Me-BLL revealed appreciably scavenged hydroxyl radical produced from the decomposition of deoxyribose in the Fenton reaction (Figure 1b). The Me-BLL showed the most effective hydroxyl radical scavenging activity with an IC 50 value of 145.08 ± 6.62 μg/mL while reference standard catechin was 37.12 ± 3.59 μg/mL.

| Iron-chelating effect
The iron-chelating effects of the Me-BLL were shown in

| Light and dark box test
In the LDB test, all the tested doses demonstrated a dose-dependent increase of time spent in the light compartment (Figure 2c). There was a notable increase in the duration of time (130.6 ± 3.72 s) for 400 mg/kg, into the light compartment, which was found significant (p < .01) compared to control (67.60 ± 2.67 s) while the value for reference drug was 177.2 ± 3.89 s (p < .01).

| Hole-board test
In HBT, the oral administration of Me-BLL in experimental animals has resulted in a significant number of head dipping (p < .01) at the doses of 200 mg/kg (53.40 ± 0.81) and 400 mg/kg (65.60 ± 1.02) in comparison to the control. Interestingly, the finding for 400 mg/kg dose was found almost similar to that of the standard drug diazepam (65.80 ± 1.62, p < .01) (Figure 2d).

| Antidepressant activity
The effects of treatment with Me-BLL on the duration of immobility times for TST and FST were presented in Figure 3a,

| Molecular docking study of anxiolytic activity
The anxiolytic molecular docking study of fifteen selected compounds was noted to interact against the potassium channel (PDB: 4UUJ) (

| Molecular docking study of antidepressant activity
The molecular docking study of antidepressant activity is summarized in Table 4. From fifteen compounds, stigmast-5-en-3.beta.  (Figure 6). Another best compound, gammasitosterol, also exhibited higher binding affinity ( Figure 6). The other 11 compounds also revealed a good docking score with the human serotonin receptor (PDB: 5I6X) receptor ( Figure S10-S13).

| Pharmacokinetic and toxicological properties
The ADME/T properties of selected eight compounds were assessed by the Lipinski's rule of five and Veber's rules. Here, cuminol F I G U R E 4 Best ranked poses and 2D interactions of (a) Cuminol (b) Thymol with urate oxidase (Uox) enzyme receptor (PDB: 1R4U) for antioxidant activity and thymol followed all the rules, whereas six other compounds violated one rule (Table 5). As all the compounds followed Lipinski's and Veber's rules except one rule, all the compounds can be predicted with good oral bioavailability. Besides, the toxicological properties of eight compounds predicted non-ames toxicity, non-carcinogenicity, weak rat toxicity except for cuminol and squalene (Table 6).

| DISCUSS IONS
Herbal medicines have been used in traditional therapies around the world for many disorders. Dietary medicinal herbs are superior antioxidant reservoirs that are safe for long-term intake (He et al., 2018). Besides, herbal medicine is turning into a feasible alternative treatment over the financially accessible synthetic drugs on neuropsychiatric management/treatment because of the lower cost, availability, and little or no adverse effects of herbal medicines (Nissen, 2010). Many well-recognized herbal products have illustrated neuropharmacological properties while mitigating anxiety and depression (Sarwar et al., 2018). that flavonoids act as free radical scavengers of many oxidizing species  and found effective in the central nervous system (Matias et al., 2016). However, the antioxidant activity of Me-BLL was evaluated employing DPPH, hydroxyl radical scavenging assay, and iron-chelating activities were done following established protocol. The overall results showed promising antioxidative effects of Me-BLL. Earlier studies suggested that the polyphenolic compounds are potent scavenger of free radicles and ROS, and their strong antioxidant activity is ascribed to the presence of ortho hydroxyl grouping, which blocks the free radical reaction and generates a hydrogen atom transfer reaction (Granato et al., 2018;Rahman et al., 2020). Moreover, a study demonstrated an elevated level of ROS produces many known mechanisms like mitochondrial deregulations, lipid cellular structures, neurotransmitter deregulations, and cellular respiration, which correlates with anxiety and depression (Rammal et al., 2010). In this regard, antioxidant  (Griebel et al., 2000). Our results revealed that the administration of Me-BLL at different doses showed a tendency to F I G U R E 6 Best ranked poses and 2D interactions of (a) Stigmast-5-en-3.   In computer-aided drug design, in silico molecular docking study is a pivotal tool that predicts the binding activity of compounds against particular proteins (Hossen et al., 2021;S. Khan et al., 2019).
Besides, the possible molecular mechanism of actions of different that none of the compounds posed a risk of Ames toxicity, acute oral toxicity, and weak rat acute toxicity and, therefore, can be considered safe.

| CON CLUS ION
The present study revealed that this edible plant has significant anxiolytic and antidepressant effects as well as antioxidant potential.
Moreover, different bioactive compounds of Me-BLL unveiled a promising avenue with a binding attraction toward different proteins in molecular docking analysis. It is noteworthy that the selected active compounds have elucidated their drug-like characteristics and safeness in ADME/T and toxicology studies. Therefore, it can be considered as an alternative food product for neuropsychiatric treatment. Further mechanistic research followed by the dose-response study is strongly recommended to elicit the neuroprotective activities of this promising plant. Note: Category-I (LD50 ≤ 50 mg/kg) and Category-III (500 mg/kg <LD50 < 5,000 mg/kg).
TA B L E 6 Toxicological properties of the selected bioactive secondary metabolites in Me-BLL

| S TUDIE S INVOLVING ANIMAL SUBJEC TS
The experimental mice were managed according to the "Guide for the Care and Use of Laboratory Animals," eight edition, USA. Animals were handled and maintained according to the recommended protocol of the Institutional Animal Ethics Committee, Department of Pharmacy, International Islamic University Chittagong, Bangladesh (reference number: P&D-147/13-19).

ACK N OWLED G EM ENTS
The authors wish to thank the Department of Pharmacy, International Islamic University Chittagong, Bangladesh, and the Laboratory of Alternative Medicine and Natural Products, University of Chittagong, Bangladesh, for providing the necessary facilities and support to conduct this research.

CO N FLI C T O F I NTE R E S T
The authors declared that they have no conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.