Scrutinizing pharmacological efficiency for Acacia auriculiformis by experimental and computational approach

The study sought to investigate the biological efficacy of methanol leave extract of Acacia auriculiformis (MEAA) via in vitro, in vivo, in silico approaches. The in vitro cytotoxicity was evaluated through brine shrimp lethality assay, and anti-inflammatory activity was determined by membrane stabilisation and protein denaturation methods (BSA and egg albumin). The in vivo antipyretic activity was examined via Brewer’s yeast induced pyrexia model. A. auriculiformis extract unveiled moderate cytotoxicity with significant anti-inflammatory efficacy (p < 0.001) compared to standard drug. This extract also exhibited dose-dependent time of paralysis and death for the worm (p < 0.001) in the anthelmintic test which was directly proportional to employed concentrations. A notable percentage of clot lysis effect (36.42 ± 1.95%, p < 0.001) was also observed for MEAA in human blood compared to control. However, this extract significantly (p < 0.05) reduced fever in a dose-dependent manner during the antipyretic experiment. Besides, in computer-aided investigations, two compounds (2,4-ditert-butylphenol and 3-hydroxy-β-damascone) revealed the best binding interaction with six proteins for cytotoxicity, inflammation, helminthic, thrombolytic and pyretic effect. Moreover, these two compounds satisfy Lipinski’s ‘Rule of Five’ and revealed drug-likeness profiles in the toxicological study. These findings disclosed that methanol leaves extract of A. auriculiformis might be a potent source for anti-inflammatory, anti-helminthic, thrombolytic and antipyretic agents.


Background
Inflammation is a biological defensive response initiated by several stimuli, including irritants, chemicals, heat, infection or any immunological reactions [1]. In general condition, inflammation shows hyperthermia and pain in the living body and aims to repair damaged tissue by inactivating deleterious stimulus or pathogen. Hence, uncontrolled acute inflammation for a prolonged period can cause drastic pain and fever in the body. These can be pathophysiology of severe diseases like cancer, rheumatoid joint pain, asthma, diabetes, multiple sclerosis, autoimmune diseases, ischemic heart disease, arthritis, atherosclerosis and Alzheimer disease [2]. However, several inflammatory mediators like prostaglandins, bioactive lipid derivative are responsible for mediation of clotting, pain vasodilation, and pyrexia [3].
Pyrexia is a complex immune biochemical reaction that initiates by producing various endogenous pyrogens as a response to inflammatory or infectious stimulation [4]. The presence of pathogens or tissue damage in the body activates the immune system which produces and releases pyrogenic cytokines. In the hypothalamus, these synthesise cytokines react with prostaglandin receptors and alter the thermoregulation system. This alteration results in increased body temperature beyond the set point of 37.2°C under the arm or axillar, 37.5-38.3°C in the anus or rectum, 37.7°C in the mouth and affects normal body functions [5]. Several antipyretic and antiinflammatory agents like paracetamol, ibuprofen and NSAIDs are widely prescribed to reduce excessive body temperature [6]. These agents mainly exert their activity by inhibiting selective and non-selective prostaglandin and cyclooxygenase enzymes. Hence, long time inhibition of these enzymes can lead to serious consequences, including iatrogenic reactions, neutropenia, GIT disorder, kidney dysfunction, liver failure and other bleeding disorders [7]. A recent alternative strategy is the inhibition of microsomal mPGES-1 (prostaglandin E synthase-1), but till now, only two inhibitors were found [8]. For these reasons, a new therapeutic approach having multifaceted pharmacological targets and less side effect has been becoming an ultimate focus for researchers all around the world.
Since the hunter-gatherer age, humans have been using natural herbs to treat various diseases [9]. Natural products have always been a vital health concern due to their phytochemicals and nutritional values as they are a decent source of antioxidants, phenolics, vitamins, fibres, minerals, bioactive metabolites [10]. In many instances of modern synthetic drug development, phytoconstituents derived from natural plants screening program assists meticulously [11]. As plant metabolites contain elevated amounts of bio-chemical compounds with effective complementary pharmacological activities, their practice in alleviating chronic diseases is becoming a great area of interest [12]. Hence, appropriate strategic research on medicinal plants to discover novel bioactive agents is inevitable. Regarding this indigenous point of view, we aimed to investigate an ethnomedicinal plant Acacia auriculiformis, in our current study.
Acacia auriculiformis is locally known as Akashmoni in Bangladesh and babul in India. A. auriculiformis belongs to Plantae kingdom, Viridiplantae subkingdom, Streptophyta infrakingdom, spermatophyte phylum, Acacia mill genus and fabaceae/leguminosae family. Traditionally, this plant is used to treat rheumatism, aches, anthelmintic, pain, sore eyes, candidiasis and conjunctivitis [13]. Australian aborigines use root decoction of this plant in the treatment of rheumatism [14]. Seeds of this plant are traditionally used in the treatment of skin ailment, rashes, itching and allergy. In Nigeria, the Ibibio community uses this plant against malaria. Several phytochemicals, including flavonoids, carbohydrates, tannins, saponins and anthocyanidins, have been reported from A. auriculiformis [13]. Isolated compounds, namely acaciaside A and B, exhibited moderate anti-microbial and anti-fungal activity via in vitro assay [15]. However, ethanolic leave extract has shown dose-dependent antiplasmodial, anti-microbial, larvicidal activity along with memory improvement and acetylcholinesterase (AChE) enzyme inhibition in rats [16,17]. Besides, Sathya et al. 2013 reported that petroleum ether extracts of empty pod and bark from this plant are potent candidates for diabetes and liver injury [18].
The plant A. auriculiformis possess several suitable medicinal properties, but until now, no study has been done reporting anti-inflammatory, anthelmintic, thrombolytic and antipyretic activities of this plant. Therefore, our present study aims to systematically explore these activities for methanol leaves extract of A. auriculiformis (MEAA). Most importantly, the current investigation aims to find out the therapeutic intervention of MEAA in mitigating inflammation by fever is the ultimate aim of our experiment. Additionally, a computational approach (molecular docking, ADME/T) was performed to predict possible insight targets of bioactive constituents of MEAA for a conceivable perspective.

Crude extract preparation
A. auriculiformis leaves were collected and identified from Rangamati region during September 19 and authenticated by a taxonomist under the accession number Anwar-0870. The leaves were washed and dried in a shaded place under 55-60°C for 6 days. Coarse powder (490 g) was obtained from dried leaves by using an electric grinder (Sahara mixer grinder, Canbara industries PVT. Ltd., Mumbai-400072). The coarse powder was soaked in an appropriate amount (1000 mL) of methanol (90%) for 7 days with occasional shaking and stirring at room temperature. The filtration was done by Whatman No. 1 filter paper in a laboratory condition 27 ± 2°C. The finale filtrate was allowed to dry and evaporate in a water bath at 45-50°C. Four gram sticky, greenish, semisolid extract was found and preserved in 4°C until further use as a methanol extract of A. auriculiformis (MEAA) [7].

Chemicals
Streptokinase vial (1,500,000 IU), loperamide and vincristine sulfate (1 mg/vial) WERE obtained from Beacon Pharmaceuticals Ltd. Bangladesh. Methanol (Merck, Germany), Tween 80 and DMSO were purchased from BDH Chemicals. Bovine serum albumin and diclofenac sodium were purchased from Square Pharmaceuticals Ltd. Normal saline solution (0.9% NaCl) was obtained from Orion Infusion Ltd. Other chemicals were procured from local sources. All the chemicals were in analytical grade.

Experimental animal
Swiss albino male mice (weight: 25-30 g) were procured from an appropriate source. All mice were housed in a laboratory for 14 days to familiarise with the standard environment. Standard guidelines such as room temperate 25 ± 2°C, relative humidity 55-60%, 12 h light/dark cycle, whereas the food pellets and water supply were maintained strictly until the experiment period. Using diethyl ether anaesthesia, the mice were euthanised after the experiment. All the mice have been slaughtered using diethyl ether anaesthesia at the end of the observation period. The ARRIVE guidelines are in compliance with for the mice model study.

In vitro study Cytotoxicity activity by brine shrimp lethality bioassay
In vitro brine shrimp lethality bioassay for MEAA was performed by the method delineated by Meyer et al. 1982 [19]. Artificial seawater was obtained by dissolving NaCl (38 g) in distilled water (1000 mL). The pH at 8.0 was attuned by adding NaOH. MEAA (5 mg/mL) concentrations (31.25, 62.5, 125, 250 and 500 μg/mL) were obtained via serial dilution. Similarly, as a standard control, serially diluted vincristine sulfate (3.13, 6.25, 12.5, 25 50 and 100 μg/mL) was used. After obtaining the concentrations, ten alive brine shrimp were placed in each tube at 25 ± 1°C. The number of alive nauplii was counted and recorded after 24 h by using the magnifying glass. The following equation used to determine the % of mortality, Here, Nt = number of nauplii taken, Na = number of nauplii alive

Anti-inflammatory test on protein denaturation inhibition by egg albumin
The anti-inflammatory activity of MEAA was studied by the egg albumin inhibiting protein denaturation method used by Guha et al. 2020 with slight modification [20]. The reaction solution (5 mL) contains egg albumin (0.2 mL), phosphate-buffered saline at pH 6.4 (2.8 mL) and MEAA extract (2 mL) varying concentration of 62.5, 125, 250 and 500 μg/mL. The exact volume of distilled water was used as a control group. In a BOD incubator (Lab-line technology, Japan), all the solutions were incubated for 15 min at 37 ± 2°C and heated for 5 min at 70°C . After cooling, the absorbance was measured at 660 nm (SHIMADZU, UV 1800) where water (vehicle) was used as a control and diclofenac sodium concentrations (31.25, 62.5, 125, 250 and 500 μg/mL) used as a reference solution. The test was carried out triplicate times, and the following equation was used to determine the percentage of protein denaturation inhibition, Abs control = absorbance of the control, Abs test = Absorbance of test Anti-inflammatory test on protein denaturation inhibition by BSA The anti-inflammatory activity of MEAA was studied by BSA inhibiting protein denaturation method formerly used by Tareq et al. 2020 with minor modifications [9]. The test mixture (0.5 mL) composed with 0.45 mL aqueous albumin (5% w/v) and 0.05 mL of MEAA extract while control mixture (0.5 mL) contain 0.45 mL aqueous albumin (5% w/v) and 0.05 mL of distilled water. The product mixture was composed of 0.05 mL of the test mixture and 0.45 mL of distilled water. The standard mixture (0.5 mL) comprises 0.05 mL of diclofenac sodium instead of distilled water. Different concentration for both test and standard mixture was obtained (125, 250, 500 and 1000 μg/mL) and pH was attuned to 6.3 by adding 1 N HCl in all concentrations. After that, all mixture was incubated for 20 min at 37°C and then kept for 30 min at 57°C. Finally, after cooling the mixture, phosphate buffer (2.5 mL) was added to each mixture and absorbance was recorded at 416 nm by using a UVvisible spectrophotometer. The test was carried out triplicate times and the following equation determined the percentage of protein denaturation inhibition, A = Absorbance of test mixture; B = Absorbance of product mixture; C = absorbance of control mixture.

Anti-inflammatory test on membrane stabilisation by hypotonic induced haemolysis
In vitro membrane stabilisation activity for MEAA was assessed by the described method Sabiu et al. 2016 [21]. The text mixture was serially diluted at 31.25, 62.5, 125, 250, 500 and 1000 μg/mL concentrations. Each concentration contains MEAA (0.5 mL), phosphate buffer (1 mL), hyposaline (2 mL) and human red blood cell (HRBC) suspension (0.5 mL). The standard mixture (diclofenac sodium) contains the same chemicals except for the extract, whereas the control mixture contains all chemicals except the standard drug and extract. All the mixtures were incubated for 30 min at 37°C. After cooling, centrifugation was for 20 min at 3000 rpm. Therefore, absorbance was taken for obtained supernatant solutions by using UV-visible spectrophotometer at 540 nm. The inhibitory haemolysis effect of MEAA was determined via the following equation Here, A = absorbance of control mixture, B = absorbance of test mixture.
In vitro anthelmintic activity on the worm Tubifex tubifex The anthelmintic activity of MEAA was evaluated by the method previously used by Adnan et al. 2018, with contemptible modifications [22]. The worms (Tubifex tubifex) were collected from aquarium shop Chittagong with 2-2.5 cm average length. The test groups contained concentrations of MEAA (5, 8 and 10 mg/mL), whereas the control group consists of distilled water and the standard group contains levamisole (1 mg/mL). Five groups were divided into five petri dish each contains randomly selected ten worms. For each aimed groups, 3 mL of the mixture was added. After that, 'time of paralysis' and 'time of death' of worms were observed and recorded carefully. When the worms stopped movement but keep vigorous shaking was considered as 'time of paralysis', and the period when the worms completely stopped movement was regarded as 'time of death'. The experiment was conducted triplet time.

In vitro thrombolytic activity via clot lysis
In vitro clot lysis activity was assessed by the described method of Prasad et al. 2006 [23]. As a test sample, MEAA crude extract (100 mg) was mixed with 10 mL distilled water and kept overnight. Streptokinase (1,500, 000 IU) was used as a stock solution. A total of 5 mL venous blood was drawn from 10 healthy and young volunteers (no history of any substance/drug used in the past 7 days). Blood retrieved from each volunteer was dispersed in pre-weighed Eppendorf tubes. Each Eppendorf containing 0.5 mL blood was incubated for 45 min at 37°C to form a clot. After incubation, layers of clot and serum were formed in each Eppendorf tubes. Serum was carefully aspirated out from each tube without disturbing the clot and the weight of tubes containing clots was measured. Therefore, 100 μL of MEAA (10 mg/mL) solution was added to each tube, whereas in the standard control tube streptokinase (SK) (100 μL), and in the control group distilled water (100 μL) was used. Then all the tubes were incubated for 90 min at 37°C. The released fluid from each tube again removed after incubation without distressing the clot. Hereafter, all the tubes weighted again to observe clot disruption. The equation described by Banu et al. 2020 [24] was followed to determine the percentage of clot lysis.

In vivo study Antipyretic test using Brewer's yeast induced pyrexia mice model
The antipyretic activity of MEAA was ascertained by the method previously used by Ahmed et al. 2019 [25]. Sixteen Swiss albino rodents were taken randomly for our current experiment and identified numerically. Then, by using a digital thermometer, the initial body temperature for all of the rodents was recorded. In the initial period, the temperature 37.25°C is considered as usual and above 37.50°C was considered as fever. Twenty percent of an aqueous suspension of Brewer's yeast (10 mL/kg, s.c.) was injected into all rodents to induce pyrexia. After pyrexia induction, all rodents were fasted overnight with only access to drink water. After 18 h, the temperature was again observed and recorded for each rodent via a digital thermometer. An increment of the temperature of more than 0.5°C confirms the induction of pyrexia [26]. Therefore, rodents that did not show temperature increment were excluded from the current experiment. Hereafter, twelve male pyrexia-induced Swiss albino mice were divided into four groups, each containing three rodents. Group I was considered negative control and received 10 mL/kg saline, whereas group II was considered a control group and received paracetamol (150 mg/kg). The remaining two groups (III and IV) were considered test groups where group III received an MEAA dose of 200 mg/kg and group IV received an MEAA dose of 400 mg/kg. After drug administration, rectal temperature was again recorded and observed for 1 h, 2 h, 3 h and 4 h periodically. Using diethyl ether anaesthesia, the mice were euthanised after the experiment. All the mice have been slaughtered using diethyl ether anaesthesia at the end of the observation period. The percentage of pyrexia reduction was determined by the following equation, Here, PiT = Pyrexia induced body temperature, Cn = temperature after 1 h, 2 h, 3 h, 4 h, NbT = normal body temperature.

ADME/T and toxicological properties analysis
The drug-like attributes (ADME/T) of the compounds were analysed based on Lipinski's rules of five by using the tool QikProp (Schrödinger v11.1) [35]. Toxicological properties were evaluated via admetSAR (Online tool) [36].

Statistical analysis
Data were uttered as mean ± SEM (standard error of the mean). The one-way statistical analysis of variance (ANOVA) was done by following post hoc Dunnett's test for multiple comparisons. In the outcomes, p < 0.05, p < 0.01 and p < 0.001 are considered as statistically significant for all cases. The analysis was carried out by using either the statistical software package for social science (SPSS, version 25.0, IBM Corporation), Prism version 8.4.2 (GraphPad Software Inc., La Jolla, CA, USA), or Microsoft Excel '13.

Effect of MEAA on brine shrimp lethality bioassay
To determine the cytotoxicity of MEAA, brine shrimp lethality bioassay was performed in the current study. Results demonstrates that the MEAA exhibited moderate cytotoxicity (LC 50 = 426.38 μg/mL) against positive control vincristine sulfate (LC 50 = 39.25 μg/mL). The lethal median dose (LC 50 ) was calculated from the regression analysis curve and the best fit line is visualised in Fig. 1.

Effect of MEAA on membrane stabilisation by hypotonic induced haemolysis
The membrane stabilisation method is widely used to determine anti-inflammatory activity due to similarities in lysosomal membranes and human red blood cell    Figure 4 outlines all the results.

Effect of MEAA on Brewer's yeast induced pyrexia mice model
Yeast-induced pyrexia model was used in our present study to identify fever relieving activity of MEAA.  Table 2.

Molecular docking for brine shrimp lethality bioassay
Human estrogen receptor (PDB ID: 1ERR) were used to perform molecular docking for brine shrimp lethality bioassay. Results show that 2-4-ditert-butylphenol gave the highest binding score (− 7.69 kcal/mol) (Fig. 6).  Table 3 and binding interactions displayed in Table 4.

Molecular docking for anthelmintic activity
Selected compounds are docked with the tubulincolchicine receptor (PDB ID: 1SA0) for anthelmintic  Values are expressed as mean ± SEM (n = 3); a p < 0.05, b p < 0.01, c p < 0.001 significantly different in comparison with control. The data were analysed by ANOVA followed by Dunnett's test  Table 3. The binding interactions are shown in Table 4.

Molecular docking for clot lysis activity
The docking result for thrombolytic activity is documented in Table 3 Table 4.

ADME/T screening for drug-likeness
Compounds compatibility as a drug molecule was investigated in the current study. According to Lipinski's 'Rules of Five', any orally administered compound must have molecular weight < 500 g/mol, hydrogen bond acceptor ≤ 10, hydrogen bond donor ≤ 5, lipophilicity value (LogP) ≤ 5. Two compounds (2-palmitoylglycerol, αmonostearin) in our study violated Lipinski's 'Rules of Five'. Further, the toxicological properties were predicted by using admetSAR online tool and confirm that none of the compounds possess carcinogenicity or AMES toxicity (Table 5). This elucidates that two derivatives (2,4ditert-butylphenol and 3-hydroxy-β-damascone) can be considered as possible lead compounds and a good candidate for the advancement of new drugs.

Discussion
Brine shrimp lethality bioassay is known as a safe, economical and effective method to evaluate the bioactivity of plant products. Although an ideal biological response depends on bioactive constituents from plant parts but analysing plant crude extract is important. A good relationship has been found for pesticidal and cytotoxic activity between this method and solid human tumours which is beneficial for natural pesticides and prescreening tests for antitumor research [19]. In this design, the higher the value of LD 50 , the lower the toxicity of the extract is and vice versa [12]. In the current study, MEAA showed an LC 50 value of 426.38 μg/mL, which is moderately toxic, and the standard control vincristine sulfate exhibited an LC 50 value of 39.25 μg/mL. These moderate toxicity might be due to the presence of triterpenoid and saponins in A. auriculiformis; therefore, it can be used as an active chemopreventive agent previously suggested by Kaur et al. 2002 [37]. Traditionally, A. auriculiformis is recommended for rheumatism, pain, itching and allergy [38]. As these causes initiate by inflammation, therefore current investigation aimed to assess the anti-inflammatory activity of MEAA. Implicit drawbacks are associated with using animal models in pharmacological research, including lack of justifications and ethical concerns [20]. Therefore, in vitro protein denaturation and membrane stabilisation methods were proposed in the ongoing study to evaluate the anti-inflammatory property. In the living body, HRBC lysis (human red blood cell) and protein denaturation are often caused by inflammation, which initiates by the excess synthesis of COX [24]. During these conditions, proteins secondary and tertiary structures      gone misplaced due to heat, strong acid or base, injurious agent, stress or organic solvent [10,39]. In the current study, at both of protein denaturation model (egg albumin, BSA) MEAA exhibited a significant percentage of inhibition (p < 0.001), which was parallel to diclofenac sodium (standard drug). Besides, in membrane stabilisation assay, dose-dependent haemolysis inhibition of MEAA (p < 0.001) was found a comparative preventative response as close to the standard diclofenac sodium (p < 0.001). An earlier finding indicated the presence of flavonoids, tannins in A. auriculiformis [13]. Studies revealed that a number of flavonoids have strong inhibitory ability against the synthesis of cyclooxygenase and prostaglandin and several other enzymes such as phospholipase A2, protein kinase C, phosphodiesterases and protein tyrosine kinases [40,41]. Besides, many investigations proposed plant metabolites like alkaloids, flavonoids, phenols and tannins possess antiinflammatory activity due to their membrane stabilisation capabilities [20,42]. Thus it can be presumed that due to the presence of flavonoids, tannins and phenols, A. auriculiformis possess significant anti-inflammatory activity. However, in-depth investigations by isolation and identification of bioactive metabolites from this plant extracts and fractions are highly recommended to reveal the exact pathway responsible for the aforementioned attributes. Around 2 billion people all around the world suffer from parasitic infections. Parasitic worm infections are known as one of the major causes for developing many complicated chronic maladies like schistosomiasis, elephantiasis and onchocerciasis [43]. Though many synthetic agents are available for anthelmintic treatment but these agents produce severe complications. Therefore, due to less or fewer side effects in plant-derived, a new agent from a natural source has become an intense interest. A. auriculiformis is widely used as an anthelmintic in various regions of the world [13]. To verify this ethnopharmacological use, we planned an experiment on the methanol leaf extract of A. auriculiformis on the worm. In our current investigation, all concentrations provoked the worm's paralysis and caused death in a dose-dependent manner. Period of paralysis and death of worms were inversely proportional to extract concentrations (p < 0.001). Numerous studies advocated that phytochemicals like alkaloids, tannins, phenolic and flavonoids are responsible for anthelmintic activity [44]. Tannins might unveil activity by uncoupling oxidative phosphorylation and interacting with the worm's energy production, leading to the death of the worm. In contrast, alkaloid acts on the CNS of worms [44]. Previously, the presence of tannins has been reported from this plant [13]. Therefore, we can assume that, due to the ubiquity of tannins in A. auriculiformis, MEAA might interact with the glycoprotein of the parasite in the gastrointestinal tract and inhibit the worm's energy production which ultimately leads to the death of the worm.
Diverse assortments of thrombolytic agents are available, which clears the network of cross-linking fibrins via triggering the plasminogen enzyme. This process makes the clot soluble and renovates blood flow in blood vessels by clearing the clogged artery, thus avoid   [45]. Therefore, MEAA could be denoted as a potential source to inhibit blood clots in the future, although this outcome is not as significant as streptokinase.
Brewer's yeast-induced pyrexia model is known as a convenient test for screening antipyretic activity of synthetic drugs along with plant phytochemicals [26]. Yeast induces in the living body causes a biochemical cascade and increase the synthesis of cytokines such as tumour necrosis factor-α (TNF-α), IL-6, interferon-α and interleukin-1β especially prostaglandins. Increased synthesis of prostaglandins alters thermoregulation in the hypothalamus [46]. Therefore, any agent that can constrain this cascade of pyrexia-inducing mediators will be marked as an antipyretic agent (Paracetamol) [47]. In the current study, administration of MEAA (200 and 400 mg/kg) exhibited reduction of rectal temperature in rodents (p < 0.05). This reduction of temperature was observed might be due to the presence of some pharmacologically active constituents in A. auriculiformis, which interfere with the synthesis of prostaglandin. However, phytochemicals, specifically steroids and flavonoids, are known as a good antipyretic agent and in the previous study presence of these phytochemicals has been observed in A. auriculiformis [13,48]. Nevertheless, it must be noted that diverse biochemical reactions take place during the synthesis of prostaglandins. Therefore, further in-depth investigation is worthwhile to regulate the exact biosynthetic pathway where the extract exerts fever relieving effects.
Computer-aided molecular docking helps to predict the molecular mechanism by ligand-target interactions and correlate the experimental data with a pharmacological response. This process plays a crucial role to bolster medical and pharmacological innovations by analysing the binding modes of active compounds against the targeted receptors. Therefore, in the present study, contemporary computer-aided molecular docking was done for previously isolated bioactive compounds via GC-MS analysis. Compounds were chosen based on Lipinski's 'Rules of Five' and further ADME/T properties were investigated. Against six targeted protein in the current study, 2-4-ditert-butylphenol unveiled promising score for inflammatory and pyretic (PDB ID: 2OYE, 6COX, 3DWW), cytotoxic (PDB ID: 1ERR) and thrombolytic (PDB ID: 1A5H) compared to other compounds. This outcome confirms that 2-4-ditert-butylphenol can directly bind with targeted enzymes and decrease enzymatic activities and reveal potential activity. Hence, in earlier, this compound expressed interaction with COX2, interleukin IL-6, IL-1b and TNF-α in RAW 264.7 cells and MCF-7 cell line activation of P53 gene has observed [49]. However, 3-hydroxy-β-damascone exhibited the best interaction score against helminthic (PDB ID: 1SA0), inflammatory and pyretic (PDB ID: 3DWW). Based on our current finding, we can conclude that due to the presence of these compounds, MEAA disclosed significant activity, and it could be a potential bioactive compound for treating several diseases.
Pharmacokinetic (ADME/T) and toxicological properties are imperative before the introduction of drugs in the commercial market. This step helps to understand the pharmacokinetic view and reduce cost during drug development. Therefore, selected compounds from methanol leaves extract of A. auriculiformis subjected to ADME/T analysis. Results exhibited that selected isolated compounds 2,4-ditert-butylphenol and 3-hydroxyβ-damascone satisfy Lipinski's 'Rule of Five' and indicate as a promising drug candidate for anti-inflammatory, antipyretic, anthelmintic and thrombolytic. In the toxicological study, selected compounds displayed non-Ames toxicity, non-carcinogenicity and none possess rat acute toxicity. Though two compounds α-monostearin and 2-palmitoylglycerol showed LD 50 values more than 5000 mg/kg in acute oral toxicity, which means that these compounds are not orally safe. Thus, in silico results in this study referred all selected isolated compounds were accountable and similarities between biological and computer-aided models results were witnessed. Therefore, this investigation gives a better understanding via correlating with pharmacokinetics and pharmacodynamics properties with the experimental outcomes for next step investigation before the clinical trial.

Conclusion
This experiment confirms that methanol leave extract of A. auriculiformis has an interesting paradox having the potentiality to contribute to the development of new antiinflammatory, antipyretic, anthelmintic and thrombolytic agents due to the presence of several secondary metabolites. In the computer-aided investigation, among all the previously isolated selected compounds, two compounds satisfied Lipinski's 'Rule of Five'. 2-4-Ditert-butylphenol unveiled significant binding interaction for antiinflammatory, antipyretic and thrombolytic activities. Moreover, 3-hydroxy-β-damascone disclosed most