
==== Front
Animal Model Exp Med
Animal Model Exp Med
10.1002/(ISSN)2576-2095
AME2
Animal Models and Experimental Medicine
2096-5451
2576-2095
John Wiley and Sons Inc. Hoboken

38979669
10.1002/ame2.12452
AME212452
AMEM-2024-0015.R2
Original Article
Regular Article
Original Article
Ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight‐mass spectrometry‐characterized extract of Aerides odorata Lour alleviates paracetamol‐induced hepatotoxicity in animal model evidenced by biochemical, molecular, and computational studies
Ahmed et al.
Ahmed A. M. Abu 1 2
Rahman Md. Atiar https://orcid.org/0000-0002-4902-8923
2 atiar@cu.ac.bd

Sharmen Farjana 1 2
Reza A. S. M. Ali 2 3
Islam Md. Shahidul 2
Rashid Md. Mamunur 2
Rafi Md. Khalid Juhani 2
Siddiqui Tanvir Ahmed 2
Ezaj Md. Muzahid Ahmed 1
Saha Srabonti 2
Uddin Md. Nazim 4
Alelwani Walla 5
1 Department of Genetic Engineering and Biotechnology University of Chittagong Chittagong Bangladesh
2 Department of Biochemistry and Molecular Biology University of Chittagong Chittagong Bangladesh
3 Department of Pharmacy International Islami University Chittagong Chittagong Bangladesh
4 Institute of Food Science and Technology, Bangladesh Council of Scientific and Industrial Research Dhaka Bangladesh
5 Department of Biochemistry, College of Science University of Jeddah Jeddah Saudi Arabia
* Correspondence
Md. Atiar Rahman, Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong 4331, Bangladesh.
Email: atiar@cu.ac.bd

09 7 2024
8 2024
7 4 10.1002/ame2.v7.4 Themed Issue: Study on Cardiovascular and Cerebrovascular Diseases 497522
12 1 2024
25 5 2024
© 2024 The Author(s). Animal Models and Experimental Medicine published by John Wiley & Sons Australia, Ltd on behalf of The Chinese Association for Laboratory Animal Sciences.
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Abstract

Background

Many kinds of orchids have significant health benefits although adequate research on their biological functions is yet to be carried out. This study investigated the paracetamol‐induced liver damage–protecting effect of epiphytic Aerides odorata methanol extract (AODE).

Methods

The protective effects of AODE were studied by analyzing its effect on liver function parameters, messenger RNA (mRNA) expression, and tissue histopathological architecture. The results were confirmed by ligand–receptor interaction of molecular docking and multitarget interaction of network pharmacological analyses.

Results

AODE significantly (p < 0.05) minimized the dose‐dependent increase in acid phosphatase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ‐glutamyl transferase, lactate dehydrogenase, and total bilirubin compared to the reference drug silymarin. Malondialdehyde level decreased, and the antioxidant genes catalase (CAT), superoxide dismutase (SOD), β‐actin, paraoxonase‐1 (PON1), and phosphofructokinase‐1 (PFK‐1) were upregulated in AODE‐treated paracetamol‐intoxicated rats. A total of 376 compounds comprising phenols and flavonoids were identified using ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight‐mass spectrometry (UPLC‐qTOF‐MS). The online toxicity assessment using SwissADME and admetSAR exhibited drug‐like, nontoxic, and potential pharmacological properties. Additionally, in silico analysis showed that isoacteoside, one of the identified compounds, exhibited the best docking score (−11.42) with the liver protein human pituitary adenylate cyclase‐1 (Protein Data Bank ID: 3N94). Furthermore, network pharmacology analysis identified the top 10 hub genes, namely AKT1 (protein kinase B), CTNNB1 (catenin beta‐1), SRC (proto‐oncogene c‐Src), TNF (tumor necrosis factor), EGFR (epidermal growth factor receptor), HSP90AA1 (heat shock protein 90α), MAPK3 (mitogen‐activated protein kinase 3), STAT3 (signal transducer and activator of transcription 3), CASP3 (caspase protein), and ESR1 (estrogen receptor 1), which are responsible for hepatoprotective activity.

Conclusion

The findings demonstrate that AODE could be a novel hepatoprotective target in drug‐induced liver damage with a further single compound–based animal study.

Hepatoprotective potential of Aerides odorata methanol extract.

Aerides odorata
animal model
mRNA expression
PCM‐induced hepatotoxicity
UPLC‐qTOF‐MS
source-schema-version-number2.0
cover-dateAugust 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:03.09.2024
Ahmed AMA , Rahman MA , Sharmen F , et al. Ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight‐mass spectrometry‐characterized extract of Aerides odorata Lour alleviates paracetamol‐induced hepatotoxicity in animal model evidenced by biochemical, molecular, and computational studies. Anim Models Exp Med. 2024;7 :497‐522. doi:10.1002/ame2.12452
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pmc1 INTRODUCTION

The liver is an essential organ that maintains the body's chemical environment by detoxifying foreign and endogenous metabolites; liver damage has been linked to oxidative stress produced by free radicals, which has become a global issue, and hepatic disease‐related fatalities are increasing at an alarming rate. Many factors are involved in frequent exposure to chemicals, medication, and pathogen infiltration (e.g., viruses and bacteria). Pharmacotherapy, liver transplantation, and surgery are the currently available treatments for liver disorders, but they have limited advantages and are accompanied by significant side effects. 1

Synthetic chemical medicines are often used to treat liver diseases, although they may have severe adverse effects such as cirrhosis, cholestatic jaundice, and increased blood transaminase levels. Paracetamol (PCM, acetaminophen, N‐acetyl‐p‐aminophenol) is often used in rat models to induce liver injury to investigate the hepatoprotective effects of natural substances. 2 Overexposure to PCM, however, has been linked to the development of inflammatory mediators, oxidative stress, imbalance of valuable biomarkers, cellular integrity, biliary tract necrosis, and hepatic necrosis. Therefore, milder and less‐expensive therapies for PCM toxicity and liver damage are needed. Polyphenols and flavonoids, which have anti‐inflammatory and antioxidative properties, are found in many herbal substances. Therefore, researchers have shown interest in the pharmacology of some herbal medicines. 3 , 4

Plants, being rich in antioxidants, can scavenge and prevent the production of free radicals. The antioxidant capacity to scavenge free radicals and reactive oxygen species (ROS) has long been linked to the mechanism of the hepatoprotective effects of certain drugs. 5 , 6 Because reports of the toxicity of synthetic antioxidants have surfaced, there has been a resurgence of interest in the search for natural antioxidants derived from plants. 7 Drugs made from plants have been shown to be nontoxic, safe, and free of serious side effects in clinical practice. 8

The Orchidaceae family is one of the largest among the most dominant angiosperm plant groups, with several species noted for their esthetic value, biological functions, or usage in various branches of the economy; they are mainly ornamental and are valued as cut flowers due to their exotic beauty and long blooming period. Although orchids are generally planted as ornamentals, most of them are used as herbal remedies and food, and for other cultural values in numerous nations and tribes of different regions of the world. 9 , 10 Besides, different bioactive compounds in orchids play a significant role in attenuating hepatotoxicity and modulating oxidative stress. 11 , 12 Furthermore, several studies have shown that natural compounds derived from edible and medicinal plants have significant antioxidant activity, which protects the liver against toxicity induced by various toxicants. 13 It has been shown that 64 medicinal plants of 88 ethnobotanical plants of Bangladesh have hepatoprotective effects in both in vivo and in vitro experiments, where only 17 species have been studied for their phytoconstituents to find the active compounds. 14 On the contrary, various kinds of orchids have bioactive phytochemicals with antioxidative properties. Dendrobium nobile, Eulophia campestris, Orchis latifolia, Vanda roxburghii, and Vanda tessellate contain alkaloids, triterpenoids, flavonoids, and stilbenoids with therapeutic benefits. Orchid extracts have been shown to possess antirheumatic, diuretic, hypoglycemic, anti‐inflammatory, antibacterial, anticonvulsive, anticarcinogenic, relaxing, antiviral, and neuroprotective properties. 15 Therefore, orchid therapy is more successful than synthetic medication; some widely used orchids are Acampe ochracea, Aerides crispum, Aerides multiflora, Aerides odorata, Anoectochilus setaceus, Apostasia nuda, Apostasia wallichii, Arundina graminifolia, Bulbophyllum lilacinum, Calanthe puberula, Cymbidium aloifolium, and Papilionanthe teres. Furthermore, A. odorata is an epiphytic orchid species with antiseptic, antibacterial, carminative, and antioxidative properties that have been used to treat a range of human ailments. Heishanzhe is a Chinese medicine that is used to treat fractures and severe injuries. 16 However, hepatoprotective effects have not been studied previously. Therefore, the aim of this study was to focus on the effects of the methanol extract of A. odorata on the hepatoprotective activity and oxidative damage in PCM‐induced rats, as well as in silico molecular docking and network pharmacology of its bioactive compounds.

2 MATERIALS AND METHODS

2.1 Chemicals and reagents

All the chemicals and reagents used in this study were of highest analytical grade. Ethanol, chloroform, isopropanol, methanol, n‐hexane, PCM, potassium persulfate (di‐potassium peroxdisulfate), and sodium acetate were obtained from Merck (Darmstadt, Germany). Ascorbic acid, catechin, Folin–Ciocalteu, gallic acid, and Trolox were purchased from Sigma‐Aldrich (St. Louis, MO, USA). All other analytical‐grade chemicals were purchased from Sigma‐Aldrich. For messenger RNA (mRNA) expression analysis, the SV total RNA isolation system, GoTaqR qPCR Master Mix (Promega, Madison, WI, USA), and GoScript (Reverse transcription system) were used.

2.2 Plant materials

The A. odorata orchids were collected from the Teknaf and Ukhiya forests of Cox's Bazar, the coastal district of Bangladesh, and taxonomical identification was performed by Professor Dr. Shaikh Bokhtear Uddin of the Department of Botany at the University of Chittagong. A voucher specimen (AM‐NPR, H101) was submitted to the Herbarium of Alternative Medicine and Natural Product Research of the Department of Biochemistry and Molecular Biology, University of Chittagong.

2.3 Extract preparation

The A. odorata orchids were cleaned with tap water, dried in the shade at room temperature, dried in an oven (40–45°C) for 5 days, and then ground into a coarse powder using a mechanical grinder (NOWAKE‐999, Japan). The resulting powder (200.0 g) was macerated in 2.0 L of methanol (99.99%) for 5 days at room temperature (25 ± 1°C) with occasional stirring at 2‐day intervals. After 5 days, the supernatant was filtered using Whatman filter paper number 1 and evaporated using a rotary evaporator (RE200, Bibby Sterling, Staffordshire, UK) at reduced pressure and temperature below 45°C. The yield of the crude extract of A. odorata (AODE) was 10.28 g (5.14% w/w).

2.4 Screening of phytochemicals

The standard procedures of Fehling's test for reducing sugars, Molisch's test for carbohydrates, Baljet test for glycoside, tests for alkaloids, frothing test for saponin, alkali test for flavonoids, FeCl3 test for tannins, and Salkowski's test for triterpenoids were performed to determine the nature of phytochemicals present in AODE. 17 , 18 , 19

2.5 Phytochemical assay of AODE using LC‐qTOF‐MS analysis

Ultra‐high‐performance liquid chromatography (UPLC) and quadrupole time‐of‐flight mass spectrometry (qTOF‐MS) were used for a quantitative phytochemical analysis of AODE. An automated Vion IMS‐qTOF‐MS (instrument software build: 2.0.0) mass spectrometer with an electrospray ionization source and an ACQUITY UPLC I‐Class system (Waters Corporation, USA) in conjunction with a Xevo G2‐S QTOF mass spectrometer was used to carry out AODE analysis using UPLC‐qTOF‐MS. Various settings were used, including a temperature source at 120°C, a high collision energy ramp start of 10.00 eV, and a high collision energy ramp finish of 40.00 eV. The scan duration of 0.200 s with an end time of 20 min was maintained ensuring the following parameters: dissolution temperature, 550°C; dissolution gas, 800 L/h; cone gas, 50 L/h; capillary voltage, 2.50 kV; and mass, 50–1000 m/z with positive polarity. For this platform, a resolution of 1.2 nm was selected. Chromatographic analysis was performed using a 150‐mm‐long CM‐A column with the filter or guard column up to 4.6‐mm internal diameter (150 × 4.6 mm for 3 μm, Waters Corporation, Franklin, MA, USA, CATL186003729). The samples were separated using water—0.1% formic acid (mobile phase A) and acetonitrile (mobile phase B) in accordance with the gradient table (Table 1). The sample and column were both set to 20 and 40°C, respectively. A photodiode‐array detector was employed (absorbance, 254 nm [two‐dimensional channel]; start wavelength, 190 nm; end wavelength, 500 nm; sample rate, 10 points/section; and pressure range, 0–18 000 psi).

TABLE 1 Gradient conditions used in the analysis.

Time (min)	Flow rate (mL/min)	Water–0.1% formic acid (composition A) (%)	Acetonitrile (composition B) (%)	Curve	
0.00	0.500	99.0	1.0	Initial	
0.50	0.500	99.0	1.0	6	
16.00	0.500	65.0	35.0	6	
18.00	0.500	0.0	100.0	1	
20.00	0.500	99.0	1.0	1	
Note: A total of 0.5 g of extract was added to methanol, and the mixture was filtered through a 0.22‐μm nylon filter before the experiment; 5 μL of the extract was injected into the analyzing column. The spectral database for organic compounds (SDBS) app's spectrum index for organic compounds was used to identify the mass fragmentations.

2.5.1 Estimation of total phenol concentration of AODE

The total phenolic concentration of AODE was determined using a well‐established technique. 20 In this experiment, 2.5 mL of Follin–Ciocalteu reagent (diluted 10 times in water) and 7.5% of Na2CO3 (2.5 mL) solution were mixed with 0.5 mL of AODE or the standard solution at various concentrations. The reactant solution was incubated at 25°C for 20 min to complete the reaction before the solution's absorbance at 760 nm was measured. The phenolic concentration and the results (represented in milligrams of gallic acid equivalent [GAE]/g of dried extracts) were determined using the gallic acid standard curve. The study was carried out in triplicate, with results reported as mean ± standard error of the mean (SEM) and values represented as GAE per gram of dried extract.

2.5.2 Total plant flavonoid estimation of AODE

Reza et al. 21 used the AlCl3 colorimetric technique to determine the total flavonoid components in AODE. The reaction was carried out using a combination of 3.0 mL of methanol, 0.2 mL of 10% AlCl3, 0.2 mL of 1 mol/L potassium acetate (CH3COOK), and 5.6 mL of distilled water, AODE, or standard solutions. The mixture's absorbance was then measured at 420 nm. The findings were represented as milligrams of quercetin equivalent (QE) per gram of dried extract, using quercetin as the standard. The experiment was carried out in triplicate, with results reported as the mean and SEM, and values represented as milligrams of QE per gram of dried extract.

2.5.3 Determination of total antioxidant capacity of AODE

A modified phosphomolybdate method was carried out to assess total antioxidant activity. 22 One milliliter of the reagent solution (0.6 mol/L of sulfuric acid, 28 mmol/L of Na₃PO₄, and 4 mmol/L of ammonium molybdate) was mixed with aliquots in screw‐cap test tubes; 1 mL of AODE sample with ascorbic acid at different concentrations (25–400 μg/mL) was used as the control. All the test tubes were incubated at 95°C for 90 min in a water bath. The absorbance rate was evaluated at 756 nm against a blank after the tubes were cooled to room temperature. All reagents were stored blank with no samples and an equivalent volume standard. The following formula was used to determine the antioxidant potential: (absorbance of the standard − absorbance of samples/absorbance of the standard) × 100 = antioxidant capacity (%).

2.6 Animal care and maintenance

Six‐ to seven‐week‐old Wistar albino rats of both sexes weighing 170–200 g were collected from the Bangladesh Council of Scientific and Industrial Research in Chittagong. The animals were maintained in standard laboratory conditions of environmental and nutritional balance at a room temperature of 23 ± 2°C, a relative humidity of 50%–60%, and a 12‐h light–dark cycle. The animals were strictly monitored throughout the experimental period to observe the unwanted environmental stresses and microbial infection by the obvious organoleptic changes in them. The animals were used and cared for according to the International Animal Ethics and Standards during the experimental process. The animals were fed a standard pellet diet collected from the International Centre for Diarrheal Disease Research, Bangladesh, Dhaka, and allowed free access to water. The animals were acclimatized for a week in polycarbonate transparent rat cages (overall size: 430 × 290 × 201 mm3 [length × breadth × height]) to ensure average growth and behavior.

They were reared under standard environmental and nutritional conditions.

2.7 Acute toxicity test for AODE

The oral acute toxicity test was performed following the Organization for Economic Co‐operation and Development's Fixed‐Dose Method (Guidelines 423) to maintain normal laboratory conditions. The rats were divided into five groups (n = 6) and treated with increasing doses of the test extract (AODE) from 200 to 2000 mg/kg body weight (bw), and 5% dimethyl sulfoxide (DMSO) was used as the control. 23 The rats were not fed after they were administered the extract. The animals were observed individually after 30 min of dosing. With special attention for the first 4 h, the animals were also observed every 24 min for the next 24 h for allergic syndromes (swelling, rash and itching of skin), and over the next 72 h for the next 14 days for behavioral changes and mortality. The efficient therapeutic dosage was confirmed to be one‐tenth of the extract's median fatal dose (Lethal dose [LD]50 > 2.0 g/kg). 24

2.8 In vivo experimental design

The PCM‐induced hepatotoxic rat models were used to assay the hepatoprotective activity of AODE. 25 The animals were split into six groups of six apiece; the normal control (NC) group, group A, received saline and 5% DMSO; the hepatic control group, group B (PCM), received 2 g/kg bw of PCM orally on the 15th day to induce toxicity; the standard control, group C (SN125), received 125 mg/kg bw of silymarin. For 14 days, groups D (AODE200), E (AODE100), and F (AODE50) received daily oral doses of 200, 100, and 50 mg/kg bw of AODE, respectively. PCM was orally administered 24 h after the last administration of the extract on the 15th day, except for group A. The animals were anesthetized using 2% halothane (category number: 1303501, Sigma‐Aldrich) 48 h after the hepatic damage was induced, and blood was collected for investigating the effects of AODE on biochemical parameters. The animals were killed by cervical dislocation, and the liver was removed and used for histopathological assay, in vivo antioxidant assay, and mRNA expression analyses. The experimental animals were handled and maintained according to the ARRIVE guidelines of the Institutional Animal Ethics Committee, Faculty of Biological Science, University of Chittagong (reference number: AERB‐FBSCU/2023‐03).

2.8.1 Biochemical analysis of animal serum

Serum was extracted from the blood by centrifuging all the blood at 3000 rpm for 15 min at 4°C, and biochemical tests were performed using commercial reagent kits (Randox Laboratories, Ireland) and AMP diagnostic kits. The manufacturer's instructions (Humalyzer 3000, Human, Germany) were followed for the whole process of analyzing acid phosphatase (ACP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ‐glutamyl transferase (GGT), total protein (TP), total bilirubin (TB), and lactate dehydrogenase (LDH).

2.8.2 Assay for antioxidant potential of AODE in liver

Determination of lipid peroxidation

Lipid peroxidation (LPO) was carried out using a modified method of Högberg et al. 26 Briefly, 1.0 g of tissue was homogenized in cold KCl (0.15 mol/L) using a handy homogenizer (FSH‐2A, YUEXIN YIQI, China). Then, 0.3 mol/L of Tris–HCL buffer (pH 7.4) and 0.02 mmol/L of sodium pyrophosphate were added to test tubes containing 0.2 mL of tissue homogenate to make a total solution volume of 2 mL. The reaction mixtures were further incubated at 37°C for 30 min in a water bath; the reaction was interrupted by adding 1 mL of 10% trichloroacetic acid (TCA), and the mixture was reincubated. Finally, 1.5 mL of thiobarbituric acid (TBA) was added after vigorous vortexing, and the reaction mixture was heated for 20 min in a boiling water bath. The experiments were repeated thrice, and the reaction mixture was observed at 532 nm. The findings of the tests were then represented as nanomole malondialdehyde (MDA) per milligram of protein reacting with thiobarbituric acid (nmol MDA/mg of protein).

Assay of AODE effect on superoxide dismutase

The effect of AODE on superoxide dismutase (SOD) activity was measured using the method described by Fridovich. 27 The liver tissues were weighed (1 g) and homogenized in cold KCl (0.15 mol/L) using a handy homogenizer (FSH‐2A, YUEXIN YIQI). SOD was estimated using sodium pyrophosphate and phenazine methosulfate. In brief, the tissue mixture was centrifuged at 15000 rpm for 60 min at 4°C. The clear supernatant (100 μL) was collected and mixed with 200 μL of 0.1 mol/L ethylenediaminetetraacetic acid (containing 0.0015% of NaCN). Then, 100 μL of 1.5 mmol/L nitroblue tetrazolium chloride (NBT) and phosphate buffer (67 mmol/L, pH 7.8) were added to make a final volume of 2.95 mL. After riboflavin was added, the absorbance was measured at 560 nm against distilled water. Then, 100 μL of 1.5 mmol/L NBT and phosphate buffer (67 mmol/L, pH 7.8) were added to a final volume of 2.95 mL. By contrasting the sample absorbance with the control, the inhibition percentage was calculated. SOD activity was evaluated (U/mg of protein) and was characterized as scavenging 50% of the superoxide anions obtained.

Assay of AODE effect on catalase activity

The catalase (CAT) activity of AODE was assayed using the method described by Beers and Sizer. 28 Briefly, 1.0 g of tissues was homogenized using cold KCl (0.15 mol/L), and 0.1 mL of the homogenate (~0.1 mg of protein) was combined with phosphate buffer (1.9 mL) and added to 1 mL of H2O2 solution. The reduction in absorption was calculated at 240 nm in 1 min with a 3‐min interval. The sample control was placed in a reference cuvette with 0.1 mL of tissue homogenate and 2.9 mL of buffer. CAT activity was calculated using the molar extinction value of 43.6. The specific activity was assessed at 25°C in millimoles of H2O2 per minute per milligram of protein sample. U/mgprotein=[(DA/min×1000×3)/43.6×mgprotein in sample]mmol/LH2O2decomposed/min/mgproteinU/mgprotein.

Assay of AODE effect on reduced glutathione

Reduced glutathione (GSH) activity was measured using the method described by Moron et al. 29 Briefly, 1.0 g of tissue was homogenized in cold KCl (0.15 mol/L), and the homogenate (500 μL) was added to 125 μL of 25% TCA, cooled on ice for 5 min, and diluted with 600 L of 5% TCA. To enable the precipitate to settle down, the tubes were centrifuged for 5 min at 3000 rpm. At this point, 350 μL of sodium phosphate buffer (0.2 mol/L, pH 8.0) and 1.0 mL of 5,5′‐Dithiobis(2‐nitrobenzoic acid), 5,5′‐(Dithiobis‐2‐nitrobenzoesäure) (DTNB) (0.6 mmol/L in 0.2 mol/L, pH 8.0 phosphate buffer) were added. The approximate yellow color reaction solution was measured at 412 nm using 150 L of supernatant, and a blank was used to compare the results. The basic curve was obtained, with GSH values ranging from 10 to 50 nmol/L. Using the normal curve, the GSH concentration of the samples was calculated and expressed as nanomole per milliliter for nanomole per milligram protein for tissue.

2.8.3 Analyzing the effect of AODE on gene expression

Isolation of total RNA

The total liver RNA was extracted using the Promega SV total RNA isolation system. Liver tissue (30 mg) was placed in a sterile microcentrifuge tube, and 175 μL of RNA lysis buffer was added to it. The tissue was homogenized using a mechanical homogenizer (Ultra‐Turrax T8, Ika‐Werke, Gmbh & Co. KG, Germany). Then, 350 μL of RNA dilution buffer (blue) was added to 175 μL of lysate. The mixture was incubated in a water bath at 70°C for 3 min. The tubes were centrifuged for 10 min at 12000–14000 g at 4°C. The supernatant was transferred to new tubes, and 200 μL of 95% ethanol was added to the clear lysate, mixing them three to four times with a pipette. The solution was placed in the spin column assembly and centrifuged at 12000–14000 g for 1 min at 4°C. Then, 600 μL of premade RNA wash solution was poured into the spin column assembly, and the mixture was centrifuged for 1 min at 12000–14000 g at 40°C. A 50‐μL freshly prepared DNase solution was administered directly to the membrane within the spin basket and incubated at 20–25°C for 15 min (5 μL of 0.09 mol/L MnCl2, 5 μL of DNase I enzyme, and 40 μL of yellow core buffer). The prepared 200 L of DNase stop solution was then added to the spin basket, which was subsequently centrifuged for 1 min at 12000–14000 g at 4°C. The basket was filled with 600 μL of the preprepared washing solution and centrifuged for 1 min at 12000–14000 g at 4°C. All the liquid from the collecting tube was removed, and 250 μL of premade RNA wash solution was added for the third time and centrifuged for 2 min at high speed. The spin basket was moved from the collecting tube to the elution tube, and 100 μL of nuclease‐free water was poured on the spin basket membrane and centrifuged for 1 min at 12000–14000 g at 4°C. Then, the spin basket was discarded, and a NanoDrop system (ND2000, Thermo Scientific, USA) was used to evaluate the RNA concentration and purity; finally, the elution tube containing pure RNA was closed and stored at −70°C.

Complementary DNA synthesis and quantitative reverse transcription PCR analysis

To synthesize complementary DNA (cDNA) from 2 μg of total RNA, a Reverse Transcription System Kit (Promega) and a SimpliAMP Thermal Cycler (Life Technologies, Applied Biosystem, USA) were utilized. In brief, total RNA was activated at 70°C for 10 min, and 20 μL of the reaction mix was added to 4 μL of reverse transcription 10× buffer, 2.5 μL of MgCl2, 0.5 μL of ribonuclease inhibitor, 1 μL of 10 mmol/L of deoxynucleotide triphosphate (dNTP) mixture, 1 μL of oligo DT, 1 ng of RNA, 1 μL of GoScript reverse transcriptase enzyme, and nuclease‐free water. The reaction mixture was then incubated for 5 min at 24°C, 60 min at 42°C, and 15 min at 70°C. For PCR amplification, the cDNA was diluted up to 50 times in nuclease‐free water. A Promega SYBR Green PCR Master Mix kit and specific primers for the enzymes SOD, CAT, paraoxonase‐1 (PON1), beta‐actin (β‐actin), and phosphofructokinase‐1 (PFK‐1) were used in the RT‐qPCR; 9 μL of Master Mix, 1.5 μL of reverse primer (10 mol/L), 1.5 μL of forward primer (10 mol/L) (Table 2), and 6 μL of cDNA were used in each 18‐μL reaction. Initial incubation was at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 51°C for 15 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. By examining the amplified product dissociation curves, the specificity of the acquired products was verified. The 2−ΔΔCT technique was used to calculate the data acquired. The target genes were then set to glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) within each sample. 30

TABLE 2 Names and sequences of the primers used for quantitative reverse transcription PCR.

Gene symbol	Gene description	Sequence (5′ → 3′)	
GAPDH*	Glyceraldehyde‐3‐phosphate dehydrogenase	F	GGTGAAGTTCGGAGTCAACGGA	
R	GAGGGATCTCGCTCCTGGAAGA	
CAT	Catalase	F	ACGAGATGGCACACTTTGACAG	
R	TGGGTTTCTCTTCTGGCTATGG	
SOD1	Superoxide dismutase 1	F	AGCTGCACCACAGCAAGCAC	
R	TCCACCACCCTTAGGGCTCA	
PON1	Paraoxonase‐1	F	TGCTGGCTCACAAGATTCAC	
R	TCAAAGCTGAGGACCTTCAAT	
PFK‐1	Phosphofructokinase‐1	F	TTACCGATCACCCTCGTTCCT	
R	TTCCCCTTAGTGCTGGGATCT	
β‐Actin	β‐Actin protein	F	GGCATCCTGACCCTGAAGTA	
R	GGGGTGTTGAAGGTCTCAAA	
Note: Asterisk refers the house keeping gene.

2.8.4 Analyzing the effect of AODE on tissue architecture

A histopathological examination revealed the impact of AODE on PCM‐induced liver damage. The tissues were cut into 3‐ to 5‐mm‐thick slices, dehydrated with ethanol, cleaned with xylene, embedded in paraffin wax, and placed on slides for microscopic examination. Several sections of the liver's cellular conditions were evaluated under an Olympus BX51 microscope, and histopathological images were obtained using an Olympus DP20 system. 31

2.9 Drug design using computer‐aided approach

2.9.1 Molecular docking simulation and analysis

To identify a suitable receptor for molecular docking considering the current study area, an extensive literature review was performed. 32 The online database RCSB Protein Data Bank (PDB, https://www.rcsb.org/) was used to import the crystal structure of adenylate cyclase (PDB ID: 3N94). 33 The resolution of the protein was 1.8 Å. LC‐qTOF‐MS was also utilized to extract the chemical structure of the major identified substances (Table 3) from AODE using PubChem (https://pubchem.ncbi.nlm.nih.gov/). 34 A total of 12 ligand structures were collected from PubChem and saved in sdf format for optimization and downstream processing (Table 3). The structures of those ligands were also drawn using ChemDraw 35 (Figure 1).

TABLE 3 The compounds and their canonical SMILES that were used for molecular docking.

Number	Compound name	Canonical SMILES	PubChem CID	
1	Hesperetin	COC1=C(C=C(C=C1)C2CC(=O)C3=C(C=C(C=C3O2)O)O)O	72281	
2	Quercetagetin‐3,4′‐dimethyl ether	COC1=C(C=C(C=C1)C2=C(C(=O)C3=C(O2)C=C(C(=C3O)O)O)OC)O	5320823	
3	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	COC1=C(C=C(C=C1)C2=C(C(=O)C3=C(O2)C(=C(C(=C3OC)OC)OC)OC)OC)OC	150893	
4	Isoacteoside	CC1C(C(C(C(O1)OC2C(C(OC(C2O)OCCC3=CC(=C(C=C3)O)O)COC(=O)C=CC4=CC(=C(C=C4)O)O)O)O)O)O	6476333	
5	3′,5‐Dihydroxy‐3,4′,7‐trimethoxy flavone	COC1=C(C=C(C=C1)C2=C(C(=O)C3=C(C=C(C=C3O2)OC)[O‐])OC)O	86289353	
6	Eupatilin	COC1=C(C=C(C=C1)C2=CC(=O)C3=C(O2)C=C(C(=C3O)OC)O)OC	5273755	
7	Casticin	COC1=C(C=C(C=C1)C2=C(C(=O)C3=C(C(=C(C=C3O2)OC)OC)O)OC)O	5315263	
8	5,7,2′,3′,4′‐Pentamethoxyflavone	COC1=C(C(=C(C=C1)C2=CC(=O)C3=C(O2)C=C(C=C3OC)OC)OC)OC	373146	
9	Erianin	COC1=C(C=C(C=C1)CCC2=CC(=C(C(=C2)OC)OC)OC)O	356759	
10	Polydatin/piceid	C1=CC(=CC=C1C=CC2=CC(=CC(=C2)OC3C(C(C(C(O3)CO)O)O)O)O)O	5281718	
11	Epicatechin gallate (epicatechin‐3‐ogallate)	C1C(C(OC2=CC(=CC(=C21)O)O)C3=CC(=C(C=C3)O)O)OC(=O)C4=CC(=C(C(=C4)O)O)O	65056	
12	2′‐Hydroxy‐3′,4′‐dimethoxy‐isoflavone‐7O‐β‐d‐glucoside	COC1=C(C(=C(C=C1)C2CC3=C(C=C(C=C3)OC4C(C(C(C(O4)CO)O)O)O)OC2)O)OC	15689656	

FIGURE 1 The two‐dimensional structures of the 12 selected ligands.

Integrated tools of the Schrödinger Suite 36 were used to prepare the proteins and ligands for dock purposes. Glide was recruited to dock the ligands with the receptors. Initially, the heteroatoms from the protein were removed along with nonessential metals, and the selenomethionines were converted into methionine. Protonation states of the receptor for the ligands were generated using Epik with a pH of 7.0 ± 2.0. Finally, the energy minimization of the receptor was performed using the OPLS3e force field. 37 The LigPrep module was used to prepare ligands. OPLS3e was also used in that case to generate all the possible significantly populated ionization states within the range of pH 7.0 ± 2.0 using Epik. Tautomers were produced for all the neutralized and ionized molecules. The Sitemap module was used to identify and verify the deep binding pockets and the surface binding pockets. Eventually, a grid box was obtained, centering the binding cleft of the receptor 3N94 for docking purposes.

2.9.2 Determination of pharmacokinetic indexes

Lipinski's rule of fives 38 and Veber's rules (topological polar surface area and number of rotatable bonds) were used to assess the absorption, distribution, metabolism, excretion, and toxicity (ADME/T) features of bioactive chemicals from AODE. QikProp (Schrödinger Release 2017‐1: QikProp, Schrödinger, LLC, New York, NY, USA) was used to examine the ADME/T properties. QikProp is a potent ADME/T prediction tool that determines whether a compound's ADME/T performance has been adequate.

2.9.3 Assaying the effect of AODE on toxicological indexes

The toxicological qualities of the chosen compounds were determined using the ADME‐T SAR online tool, which is a significant issue during the development of novel medications. 39 Therefore, the aim of this research was to assess toxicity as well as carcinogenic characteristics, acute oral toxicity, and acute rat toxicity.

2.9.4 Network pharmacological analysis

Construction of bioactive compound–multitarget protein network

We used the SwissTargetPrediction tool to obtain the associated possible bioactive targets (http://www.swisstargetprediction.ch/). 40 The canonical simplified molecular input line entry system (SMILES) structures of the selected compounds were coded into the tool. The compound targets with no relationship with the compound–protein interactions were not considered for future investigation. The threshold of compound–target interaction probability was >0.1.

Construction of protein–protein interaction network of the predicted genes

Using the STRING database (https://string‐db.org/cgi/input.pl; STRING‐DB, version 11.0) search engine, a protein–protein interaction (PPI) network was formed for the expected genes. 41 The Cytoscape plugin cytoHubba was used to determine the rank of the target proteins based on the strength of connections in the PPI network. 42 To form a PPI network, the gathered protein interaction information for each target protein was loaded into Cytoscape, version 3.6.1. 43

Analysis of the target proteins' pathway enrichment using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases

The Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/), version 6.8, was used to determine the function of target proteins that interact with the active components in gene function and signaling pathway. 44 The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were strongly linked to the expected and identified genes (Kanehisa et al., 2017). The KEGG pathway enrichment and Gene Ontology (GO) function of the target proteins were examined. Additionally, the target proteins for the KEGG pathways, cellular components (CC), molecular function (MF), and biological process (BP) were specified. The false discovery rate value <0.05 was considered statistically significant.

2.10 Statistical analysis

The data were analyzed using one‐way analysis of variance (ANOVA) followed by Dunnett's test using the Statistical Package for Social Sciences (SPSS, version, 22.0, IBM Corporation, New York, USA) and GraphPad Prism Data Editor for Windows, version 8.4.3 (GraphPad Software Inc., San Diego, CA, USA). All the values were expressed as mean ± SEM. p‐Values (c p < 0.05, b p < 0.01, and a p < 0.001) were considered statistically significant.

3 RESULTS

3.1 Qualitative phytochemical screening

The qualitative phytochemical screening of AODE detected the presence of different secondary metabolites such as alkaloids, carbohydrates, flavonoids, glycosides, phenols, steroids, and tannins (Table S1).

3.2 Quantitative phytochemical status

3.2.1 Phytometabolites from LC‐qTOF‐MS analysis

The methanolic extracts of AODE were used in LC‐qTOF‐MS analysis to determine the phytochemical characteristics of the entire plant. The experiments differentiated flavonoids, phenols, and steroid compounds, and glycosides (Table 4). The chromatogram is shown in Figure 2. Flavonoids made up most of the compounds, including 3,5,6,7,8,3′,4′‐heptemethoxyflavone (R/t 3.59) and irisolidone (R/t 6.98). Major polyphenols identified in AODE were darendoside A (R/t 3.46), ciwujiatone (R/t 9.80), and moupinamide (retention time [R/t] 10.46). Large concentrations of valuable antioxidative compounds such as prenylated (+)‐polydatin (R/t 5.68), apocynin B (R/t 5.71), nobilin B (R/t 5.72), kushenol V (R/t 7.73), isosilybin (R/t 10.77), lusianthridin (R/t 11.63), tribulusamide A (R/t 12.58), 5,7‐dihydroxy‐6‐methyl‐3‐(4′‐hydroxybenzyl)chromone (R/t 14.59), and 2,5,7‐trihydroxy‐6,8‐dimethyl‐3‐(4′‐methoxybenzyl)chroman‐4‐one (R/t 16.63) were present in AODE.

TABLE 4 First 100 identified phytoconstituents in AODE using LC‐qTOF‐MS.

Number	Compound name	Formula	Identification status	Observed neutral mass (Da)	Observed m/z	Mass error (mDa)	Mass error (ppm)	Observed RT (min)	Response	Adducts	Observed collision cross section (CCS) (Å2)	Total fragments found	
1	(3R)‐2′,3′,7‐Trihydroxy‐4′‐methoxyisoflavanone	C16H14O6	Identified	302.0795	309.0949	0.4	1.4	1.92	1224	+Li	164.99	0	
2	Hesperetin	C16H14O6	Identified	302.0795	309.0949	0.4	1.4	1.92	1224	+Li	164.99	0	
3	3,4‐Dihydroxybenzamide	C7H7NO3	Identified	153.0425	154.0497	–0.1	−0.8	2.23	1192	+H	134.04	0	
4	Divaricatol	C17H18O7	Identified	334.1054	341.1209	0.2	0.5	2.70	1764	+Li	181.31	0	
5	Quercetagetin‐3,4'dimethyl ether	C17H14O8	Identified	346.0691	353.0846	0.3	0.8	2.75	1565	+Li	178.93	0	
6	Decaffeoylacteoside	C20H30O12	Identified	462.1751	485.1644	1.4	2.9	3.09	1099	+Na	203.28	0	
7	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1406	433.1479	−1.4	−3.3	3.44	1746	+H	202.60	0	
8	Darendoside A	C19H28O11	Identified	432.1641	455.1533	1.0	2.1	3.46	1982	+Na	196.33	0	
9	Osmanthuside H	C19H28O11	Identified	432.1641	455.1533	1.0	2.1	3.46	1982	+Na	196.33	0	
10	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1398	433.1470	−2.3	−5.2	3.59	2449	+H	207.03	0	
11	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1399	433.1472	−2.1	−4.8	3.75	1921	+H	196.67	0	
12	Isoacteoside	C29H36O15	Identified	624.2045	625.2118	−0.9	−1.4	3.77	1085	+H	237.67	0	
13	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1406	433.1479	−1.4	−3.3	3.95	2192	+H	198.43	0	
14	7‐Hydroxy‐3,5,6,8,3′,4′‐hexamethoxyflavone	C21H22O9	Identified	418.1274	425.1428	1.0	2.3	4.05	2187	+Li	189.08	0	
15	3′,5‐Dihydroxy‐3,4′,7‐trimethoxy flavone	C18H16O7	Identified	344.0902	351.1056	0.6	1.6	4.14	2580	+Li	174.46	0	
16	Eupatilin	C18H16O7	Identified	344.0902	351.1056	0.6	1.6	4.14	2580	+Li	174.46	0	
17	Noririsflorentin	C19H16O8	Identified	372.0852	379.1007	0.7	1.8	4.18	1599	+Li	190.61	0	
18	2′‐Hydroxyisoophiopogonone A	C18H14O7	Identified	342.0746	349.0900	0.6	1.8	4.59	1048	+Li	172.73	0	
19	Kuwanon A	C25H24O6	Identified	420.1535	421.1608	−3.8	−9.0	4.59	1445	+H	195.65	0	
20	Dihydrooxyresveratrol	C14H14O4	Identified	246.0893	253.1048	0.1	0.4	4.64	2631	+Li	152.22	0	
21	Decaffeoylacteoside	C20H30O12	Identified	462.1752	485.1644	1.5	3.1	4.74	1947	+Na	202.65	0	
22	Casticin	C19H18O8	Identified	374.1012	381.1167	1.0	2.7	4.83	2031	+Li	181.06	0	
23	10‐O‐Methylprotosappanin B	C17H18O6	Identified	318.1108	325.1263	0.5	1.4	4.88	2967	+Li	172.15	0	
24	4‐O‐Methylsappanol	C17H18O6	Identified	318.1108	325.1263	0.5	1.4	4.88	2967	+Li	172.15	0	
25	Ledebouriellol	C20H22O7	Identified	374.1378	381.1532	1.2	3.2	4.89	1574	+Li	191.61	0	
26	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1439	439.1594	1.9	4.3	5.04	1148	+Li	201.16	0	
27	5,7,2′,3′,4′‐Pentamethoxyflavone	C20H20O7	Identified	372.1220	373.1293	1.1	3.0	5.13	1337	+H	194.89	2	
28	Torachrysone‐8‐O‐β‐d‐glucopyranoside	C20H24O9	Identified	408.1432	431.1324	1.2	2.7	5.14	2087	+Na	205.93	0	
29	Chrysotoxine	C18H22O5	Identified	318.1464	325.1618	−0.4	−1.1	5.19	4218	+Li	172.33	0	
30	Erianin	C18H22O5	Identified	318.1464	325.1618	−0.4	−1.1	5.19	4218	+Li	172.33	0	
31	Darendoside A	C19H28O11	Identified	432.1632	455.1525	0.1	0.2	5.23	11 931	+Na, +K	202.17	1	
32	Osmanthuside H	C19H28O11	Identified	432.1632	455.1525	0.1	0.2	5.23	11 931	+Na, +K	202.17	1	
33	Carthamidin	C15H12O6	Identified	288.0640	295.0795	0.7	2.2	5.28	1793	+Li	166.16	0	
34	Kuzubutenolide A	C23H24O10	Identified	460.1352	461.1425	−1.7	−3.8	5.32	3427	+H	196.12	1	
35	3′,4′,5′,5,7,8‐Hexamethoxy flavone	C21H22O8	Identified	402.1322	403.1395	0.8	1.9	5.32	1610	+H	200.60	0	
36	Moracin M‐3′‐O‐β‐d‐glucopyranoside	C20H20O9	Identified	404.1118	411.1272	1.0	2.5	5.34	1373	+Li	187.12	1	
37	Kuzubutenolide A	C23H24O10	Identified	460.1349	461.1422	−2.0	−4.4	5.42	3727	+H	195.59	2	
38	3′,4′,5′,5,7,8‐Hexamethoxy flavone	C21H22O8	Identified	402.1314	403.1386	−0.1	−0.2	5.43	1461	+H	202.12	0	
39	Nobilin B	C17H20O6	Identified	320.1254	327.1409	−0.6	−1.8	5.52	3130	+Li, +H	172.92	0	
40	Apocynin B	C24H20O10	Identified	468.1065	475.1220	0.9	1.8	5.59	1221	+Li	199.30	0	
41	Dihydrooxyresveratrol	C14H14O4	Identified	246.0893	253.1048	0.1	0.5	5.60	1728	+Li	155.12	0	
42	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1455	439.1610	3.5	8.0	5.63	1489	+Li	197.05	0	
43	Polydatin	C20H22O8	Identified	390.1322	397.1477	0.8	2.0	5.68	1963	+Li	194.27	0	
44	Apocynin B	C24H20O10	Identified	468.1069	475.1224	1.3	2.7	5.71	1097	+Li	197.52	0	
45	Nobilin B	C17H20O6	Identified	320.1259	327.1414	0.0	−0.1	5.72	1762	+Li	166.83	0	
46	Obtustyrene	C16H16O2	Identified	240.1154	247.1309	0.4	1.6	5.74	2058	+Li	199.92	0	
47	5,7,2′,3′,4′‐Pentamethoxyflavone	C20H20O7	Identified	372.1192	373.1264	−1.7	−4.7	5.77	2481	+H	192.54	0	
48	Epicatechin gallate (epicatechin‐3‐O‐gallate)	C22H18O10	Identified	442.0937	449.1092	3.7	8.3	5.84	1535	+Li	267.00	1	
49	Meliadanoside A	C16H24O10	Identified	376.1390	377.1462	2.0	5.3	5.86	4442	+H, +Na	187.49	2	
50	Nobilin B	C17H20O6	Identified	320.1278	327.1432	1.8	5.4	5.89	1463	+Li	172.72	1	
51	N‐Methyltyramine	C9H13NO	Identified	151.0998	152.1071	0.1	0.6	5.92	1112	+H	140.56	0	
52	Ledebouriellol	C20H22O7	Identified	374.1370	381.1524	0.4	1.1	5.94	3594	+Li	187.15	2	
53	Irisflorentin	C20H18O8	Identified	386.0975	387.1048	−2.6	−6.8	6.01	6057	+H	195.26	0	
54	5,7,2′,3′,4′‐Pentamethoxyflavone	C20H20O7	Identified	372.1187	373.1260	−2.2	−5.9	6.06	2570	+H	190.15	0	
55	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1422	439.1577	0.2	0.5	6.07	4056	+Li	196.04	1	
56	Glabrol	C25H28O4	Identified	392.2016	393.2089	2.8	7.2	6.10	7685	+H	180.26	0	
57	2,3‐Dihydroiriegnin	C18H18O8	Identified	362.1009	369.1163	0.7	2.0	6.19	1486	+Li	186.71	1	
58	7‐Hydroxy‐5,3′,4′‐trimethoxy flavone	C18H16O6	Identified	328.0949	329.1022	0.3	0.8	6.19	1097	+H	181.42	1	
59	Irisflorentin	C20H18O8	Identified	386.0974	387.1046	−2.8	−7.3	6.19	6067	+H	191.87	1	
60	7‐Hydroxy‐3,5,6,8,3′,4′‐hexamethoxyflavone	C21H22O9	Identified	418.1275	425.1430	1.1	2.6	6.27	1405	+Li	192.74	0	
61	Stilbostemin D	C16H18O3	Identified	258.1265	265.1420	0.9	3.6	6.35	1378	+Li	160.00	0	
62	3,5,2′,4′‐Tetrahydroxystilbene	C14H12O4	Identified	244.0735	251.0890	0.0	−0.1	6.37	1342	+Li	154.13	0	
63	Norkurainol	C25H28O7	Identified	440.1839	463.1731	0.4	0.8	6.54	1025	+Na	220.98	1	
64	2′‐Hydroxy‐3′,4′‐dimethoxy‐isoflavone‐7‐O‐β‐d‐glucoside	C23H28O10	Identified	464.1689	487.1581	0.7	1.4	6.69	1995	+Na	204.24	4	
65	2,5,7‐Trihydroxy‐6,8‐dimethyl‐3‐(3′,4′‐methylenedioxybenzyl)chroman‐4‐one	C19H18O7	Identified	358.1046	365.1200	−0.7	−1.9	6.71	3062	+Li	177.01	0	
66	Corymbosin	C19H18O7	Identified	358.1046	365.1200	−0.7	−1.9	6.71	3062	+Li	177.01	0	
67	4′‐Methylpinosylvin	C15H14O2	Identified	226.0977	249.0869	−1.7	−6.8	6.81	1581	+Na	154.22	0	
68	(3R,4S)‐4′,7‐Dimethoxy‐3′‐deoxysappanol	C18H20O5	Identified	316.1314	323.1469	0.3	1.0	6.90	3415	+Li	170.93	2	
69	2′‐Hydroxy‐7,3′,4′‐trimethoxy‐isoflavone	C18H20O5	Identified	316.1314	323.1469	0.3	1.0	6.90	3415	+Li	170.93	3	
70	Irisolidone	C17H14O6	Identified	314.0795	321.0950	0.5	1.5	6.98	3287	+Li	178.58	0	
71	Stilbostemin D	C16H18O3	Identified	258.1267	265.1422	1.1	4.3	6.99	2135	+Li	159.09	0	
72	1,2,3,5‐Tetramethoxyxanthone	C17H16O6	Identified	316.0951	323.1106	0.4	1.3	7.00	5209	+Li	175.76	1	
73	Caesalpins J	C17H16O6	Identified	316.0951	323.1106	0.4	1.3	7.00	5209	+Li	175.76	2	
74	3′,5′,β‐Trihydroxy‐3,4,4′,α‐tetramethoxychalcone	C19H20O8	Identified	376.1141	377.1214	−1.7	−4.6	7.07	2214	+H	194.92	0	
75	Blestrianol D	C29H24O5	Identified	452.1659	453.1732	3.6	7.9	7.08	1985	+H	198.31	0	
76	Thannilignan	C19H22O5	Identified	330.1473	337.1627	0.5	1.6	7.08	2438	+Li	174.98	0	
77	5‐Hydroxyauranetin	C20H20O8	Identified	388.1147	389.1220	−1.1	−2.8	7.17	1276	+H	191.33	1	
78	Methyl‐5‐O‐caffeoylquinate	C17H20O9	Identified	368.1117	391.1010	1.0	2.6	7.18	1270	+Na	197.18	0	
79	3′,4′,5′,5,7,8‐Hexamethoxy flavone	C21H22O8	Identified	402.1294	403.1366	−2.1	−5.2	7.19	1292	+H	278.31	0	
80	3,5,6,7,8,3′,4′‐Heptemethoxyflavone	C22H24O9	Identified	432.1408	433.1481	−1.2	−2.9	7.19	1625	+H	205.92	1	
81	2,7‐Dihydroxy‐4‐methoxyphenanthrene‐2‐O‐glucoside	C21H22O8	Identified	402.1294	403.1366	−2.1	−5.2	7.19	5925	+H	198.26	3	
82	2,5,7‐Trihydroxy‐6,8‐dimethyl‐3‐(4′‐methoxybenzyl)chroman‐4‐one	C19H20O6	Identified	344.1262	351.1417	0.2	0.7	7.20	1389	+Li	176.52	1	
83	Octahydrocurcumin	C21H28O6	Identified	376.1892	383.2046	0.6	1.5	7.21	2610	+Li	187.47	0	
84	5‐Hydroxyauranetin	C20H20O8	Identified	388.1162	395.1316	0.4	0.9	7.32	2825	+Li	185.22	1	
85	3′,4′,7‐Tribenzylepisappanol	C37H34O6	Identified	574.2327	575.2399	−2.9	−5.0	7.36	1078	+H	230.30	1	
86	4‐O‐Methylsappanol	C17H18O6	Identified	318.1112	341.1004	0.8	2.5	7.51	1160	+Na	186.76	2	
87	Tilianin	C22H22O10	Identified	446.1192	447.1265	−2.1	−4.7	7.53	1997	+H	206.95	2	
88	Dendrocandin A	C17H20O5	Identified	304.1283	305.1356	−2.8	−9.1	7.60	2522	+H	171.30	0	
89	Nobilin C	C18H22O6	Identified	334.1409	335.1482	−0.7	−2.1	7.60	1292	+H	173.78	2	
90	Sanggenon H	C20H18O6	Identified	354.1105	355.1178	0.2	0.6	7.67	1256	+H	190.38	2	
91	Polydatin	C20H22O8	Identified	390.1312	413.1204	−0.3	−0.7	7.68	7082	+Na	207.71	1	
92	Morusinol	C25H26O7	Identified	438.1634	445.1789	−4.4	−10.0	7.71	1329	+Li	199.52	2	
93	Kushenol V	C21H22O7	Identified	386.1378	387.1451	1.3	3.3	7.73	1162	+H	198.62	0	
94	Irisflorentin	C20H18O8	Identified	386.1007	393.1162	0.5	1.4	7.78	1485	+Li	194.88	0	
95	Renifolin	C18H24O7	Identified	352.1507	353.1580	−1.5	−4.3	7.82	1894	+H	180.16	0	
96	Polydatin	C20H22O8	Identified	390.1321	413.1213	0.6	1.5	7.87	4407	+Na	203.76	2	
97	Sanggenon A	C25H24O7	Identified	436.1527	443.1681	0.5	1.1	7.88	1670	+Li	203.15	2	
98	Dihydroresveratrol	C14H14O3	Identified	230.0944	237.1099	0.1	0.4	8.04	1882	+Li	150.31	0	
99	Ciwujiatone	C22H26O9	Identified	434.1605	435.1678	2.8	6.5	8.07	1337	+H	200.70	2	
100	(6‐Methoxy‐2‐[2‐3′‐methoxy‐4′‐hydroxyphenyl)ethyl]chromone)A	C19H18O5	Identified	326.1161	327.1234	0.7	2.0	8.14	2752	+H	178.00	3	
Abbreviations: LC‐qTOF‐MS, ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight‐mass spectrometry; RT, retention time.

FIGURE 2 Analysis using UPLC‐QTOF‐MS (ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight‐mass spectrometry). Aerides odorata extract liquid chromatography‐mass spectrometry (LC–MS).

3.2.2 Total antioxidative capacity, total phenol concentration, and total flavonoid concentration of AODE

The quantitative analysis of total antioxidant capacity, total plant phenol concentration, and flavonoid concentration is presented in Table 5. The antioxidant capacity of AODE was found to be 251.76 ± 20.55 mg of ascorbic acid (AA) per gram of dried extract. The phenol concentration in AODE was 53.73 ± 2.11 mg of GAE per gram of dried extract, the highest concentration of flavonoid was found to be 108.17 ± 4.69 mg of QE per gram, and 43.34 ± 1.51 mg catechin (CATE) per gram was recorded from the dried extract of AODE.

TABLE 5 Quantitative analysis of antioxidant‐relevant phytochemicals of AODE.

Antioxidative indices	AODE	
Total phenol concentration (mg QE/g crude extract)	53.73 ± 2.11	
Total flavonoid concentration (mg GAE/g crude extract)	108.17 ± 4.69	
Total antioxidant capacity (mg AA/g crude extract)	251.76 ± 20.55	
Proanthocyanidin concentration mg CATE/g plant extract	43.34 ± 1.51	
Abbreviations: AA, ascorbic acid; AODE, Aerides odorata methanol extract; GAE, gallic acid equivalent; QE, quercetin equivalent.

3.3 Effect of AODE on acute toxicity

The analysis showed that oral administration of AODE at the maximum dose of 2000 mg/kg bw did not exhibit any toxicity or anomalies. No abnormal organoleptic changes were recorded throughout the experimental period.

3.4 Effect of AODE on physiological changes

Table 6 presents how AODE therapy affected the animals' physiological changes. After PCM induction, the weights of rat liver were shown to be considerably (p < 0.001) different from those of the NC group. On the contrary, there were no discernible variations in liver weights between the NC group and the AODE‐treated groups. The relative organ weights between the NC and SN125 (silymarin 125 mg/kg bw, reference control) groups and the AODE‐treated group were not statistically different, whereas PCM induction had a significant (p < 0.001) influence on changes in liver and organ weights.

TABLE 6 Effect of AODE on physiological changes in paracetamol‐induced rats.

Treatment group	Initial body weight	Final body weight	Body weight changes (%)	Liver weight	Relative organ weight	
NC	181.00 ± 1.73	198.00 ± 2.00a	9.39 ± 0.56	6.58 ± 0.82	3.33 ± 0.49	
PCM500	194.67 ± 1.15	175.33 ± 1.15a	9.93 ± 0.57a	11.04 ± 0.16a	6.28 ± 0.10b	
AODE00	204.33 ± 0.59a	191.67 ± 3.21	6.63 ± 1.72c	6.54 ± 0.65ns	3.20 ± 0.32ns	
AODE100	160.27 ± 1.62a	152.67 ± 2.52	4.99 ± 0.76a	7.02 ± 1.17ns	4.38 ± 0.76ns	
AODE50	172.93 ± 2.61b	167.07 ± 2.69	3.51 ± 0.17a	6.75 ± 1.82ns	3.92 ± 1.04ns	
SN125	191.00 ± 2.00	204.67 ± 0.58a	7.16 ± 1.17#	6.81 ± 0.69#	3.3 ± 0.33#	
Note: The mean ± SEM (standard error of the mean) of the seven animals in each group is used to calculate the values. Analysis of variance (ANOVA) was used for statistical analysis, followed by Dunnett's t‐test. When compared to the paracetamol control; “a,” “b,” and “#” indicate the significant difference at p < 0.01 and p < 0.001 levels, and “ns” denotes nonsignificance. NC denotes normal control (distilled water), PCM denotes hepatic control (paracetamol 500 mg/kg bw [body weight]), and SN125 denotes silymarin 125 mg/kg bw.

3.5 Impacts of AODE on biochemical indexes

Figure 3A–H shows that when PCM was administered over an extended period, the serum ACP level significantly increased compared to the NC (p < 0.001). Additionally, the ACP levels of both the SN and AODE 200‐mg groups were significantly (p < 0.001) minimized compared to the PCM‐induced group. The effect of AODE 100‐mg and AODE 50‐mg groups was found to be nonsignificant compared to the PCM‐treated group. The AODE 200‐mg and AODE 100‐mg groups as well as the SN 125‐mg group significantly (p < 0.001) reduced the levels of AST, ALP, ALT, and LDH compared to the PCM group. Furthermore, treatment with SN 125‐mg silymarin and 200‐mg/kg AODE significantly (p < 0.001) increased the TP level compared to PCM. The AODE treatment significantly (p < 0.001) and dose dependently improved TB and GGT.

FIGURE 3 Effect of AODE on serum (A) ACP (acid phosphatase) and (B) ALT (alanine aminotransferase), (C) AST (aspartate aminotransferase), (D) ALP (alkaline phosphatase), (E) GGT (glutamyl transferase), (F) LDH (lactate dehydrogenase), (G) TB (total bilirubin), and (H) TP (total protein) after PCM (paracetamol) treatment. Pretreated extracts decreased the serum ACP, AST, ALP, GGT, LDH, and TB, and increased the TP activities. Results are represented as the mean ± SD (standard deviation), where n = 6.

3.6 Effects of AODE on antioxidant enzymes

The analysis results indicated a substantial oxidative shift with a spike in LPO as shown by an increase in MDA (Figure 4A) level in the PCM‐treated group, whereas treatment with AODE (200 mg/kg) and silymarin (125 mg/kg) (p < 0.001) significantly decreased LPO. Apart from these, 100 mg/kg of AODE considerably (p < 0.01) decreased LPO; however, 50 mg/kg is not significant when all treatments are compared to PCM. Endogenous antioxidative enzymes such as CAT (Figure 4B) and SOD (Figure 4C) were markedly reversed in a dose‐dependent manner with the administration of 100 mg/kg of AODE and 125 mg/kg of silymarin. Furthermore, AODE (100 and 200 mg/kg bw) and silymarin (125 mg/kg bw) substantially exacerbated (p < 0.001) the PCM‐induced decrease in GSH activity (Figure 4D), whereas AODE (50 mg/kg) had no impact.

FIGURE 4 Changes in malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and reduced glutathione (GSH) levels because of AODE treatment. Reference standard drug, SN125 (silymarin 125 mg/kg bw [body weight]); NC, normal control, distilled water; PCM, hepatic control induced by paracetamol. All medications were taken orally. Compared to normal and hepatic control, values for six animals in each group are presented as mean ± SD (standard deviation). (A) Effect of AODE on MDA concentration; (B) Effect of AODE on CAT content; (C) Effect of AODE on SOD content; (D) Effect of AODE on GSH content.

3.7 Effect of AODE on regulation of CAT , SOD , PON1 , β‐actin, and PFK‐1

The investigation on the hepatoprotective effects of AODE on PCM‐stimulated liver damage in rats quantitatively assessed the relative mRNA expression levels of key antioxidative and metabolic genes (Figure 5). The study utilized six animals per experimental group, and the treatments included NC, PCM‐induced hepatic control, silymarin at 125 mg/kg (SN125), and AODE (250 mg).

FIGURE 5 Effect of AODE on CAT (catalase), SOD (superoxide dismutase), β‐actin, PON1, and PFK (phosphofructokinase) mRNA (messenger RNA) expression levels in paracetamol (PCM)–stimulated rat liver. All medications were given orally. PCM, hepatic control by paracetamol; NC, normal control, distilled water; reference standard drug, SN125 (silymarin 125 mg/kg). For six animals in each group, values are expressed as mean ± SD (standard deviation); two‐way ANOVA (analysis of variance) and Dunnett's multiple comparisons test were performed. Different letters “a,” “b,” and “c” denote different degrees of significance in relation to PCM (α).

The analysis revealed that AODE treatment resulted in a pronounced increase in the mRNA expression of CAT and SOD, which were significantly higher compared to the PCM group, with the differences being statistically significant (p < 0.05). This suggests that AODE possesses potent antioxidant properties that may contribute to its hepatoprotective action. In contrast, the expression of β‐actin remained unchanged across all treatment groups, serving as a reliable internal control.

Furthermore, the mRNA expression levels of PON1 and PFK were significantly elevated in the AODE‐treated group compared to the PCM group. The enhancement in PON1 expression indicates an increase in the detoxification capacity, whereas the upregulation of PFK suggests an improvement in the metabolic efficiency of the liver after AODE treatment.

3.8 Effects of AODE on liver tissue architecture

Tissue architecture of the liver segment of the control group was observed to have the typical histological architecture (Figure 6A), but the PCM‐induced group exhibited several architectural distortions. The PCM‐induced liver exhibited necrotic alterations and hepatocyte degradation. Additionally, many abnormalities such as edema, necrosis, degeneration, and apoptosis were observed (Figure 6B), whereas the liver segment of rats treated with silymarin exhibited a significant decrease in various abnormalities (Figure 6C). Pretreatment with AODE liver restored its various structural defects in a dose‐dependent manner, as observed in Figure 6E,F. The maximum dose (200 mg/kg) restored many abnormalities such as degenerative and centrilobular necrosis, single‐cell necrosis, and apoptosis more effectively than the medium and low doses (100 and 50 mg/kg), which is nearly similar to the reference medicine silymarin.

FIGURE 6 Effect of AODE on the architecture of liver tissue shown in histological assay (40× at scale bar = 500 μm, H&E [hematoxylin and eosin]). (A) Normal liver tissue sections with no damage to the hepatic cell architecture; (B) hepatic control confirms the hepatocellular damage through a variety of inflammations, such as necrosis, sinusoidal dilation, apoptosis, hemorrhage, and dilatation in the central vein with diffused Kuppfer cell proliferation between hepatocytes; (C) a drug called silymarin, which is used to treat liver damage, was used as tissue sections with less inflammation, centrilobular necrosis, and sinusoidal dilution, demonstrating a positive regeneration of liver cells from injury in the treatment groups. (D) AODE 200, (E) AODE 100, and (F) AODE 50 mg/kg bw (body weight). The liver tissues were embedded in paraffin cubes, cut with a microtome, and then stained with H&E. The arrow indicates the central lobular position of hepatic cells. BS, Blood sinusoids; DG, degenaration; SN, single cell necrosis; IF, inflammation; CV, central vein; AP, apoptosis.

3.9 Molecular docking studies

The receptor was refined and minimized and was ready to be docked with the desired ligands. After ligand optimization, we found ~123 tautomers of the 12 ligands, and each of them was equally qualified for the docking study. To continue with the docking, we maintained an extra precision mode for intensive analysis of the results with proper details. The docking results are presented in Table 7, where the first tautomer of the ligand CID‐6476333 exhibits the highest docking score of −11.42 kcal/mol. The other tautomers had a range between −11.16 and −2.47 kcal/mol. To avoid complexity, here we discussed only the CID‐6476333 tautomer‐1‐docked complex with the receptor.

TABLE 7 Docking scores and relevant indexes for the selected compounds for the study.

Ligand	GScore	Dock score	Lipophilic EvdW	PhobEn	PhobEn PairHB	HBond	Electro	Sitemap	LowMW	Penalties	Expos penal	Rot penal	Epik state penalty	Activity	
6476333	−11.42	−11.42	−4.06	−0.3	0	−5.24	−1.89	−0.12	0	0	0.05	0.14	0	45.47	
6476333‐2	−11.16	−11.16	−2.37	0	0	−6.96	−2	−0.01	0	0	0.04	0.14	0	40.35	
6476333‐3	−10.76	−10.76	−2.83	−0.3	0	−6.49	−2	0	0	0.19	0.47	0.2	0	43.28	
6476333‐4	−10.44	−10.44	−3.95	0	0	−4.91	−1.59	−0.14	0	0.01	0	0.14	0	50.16	
6476333‐5	−10.38	−10.38	−2.89	−0.3	0	−6.17	−1.91	0	0	0	0.74	0.14	0	44.08	
6476333‐6	−10.18	−10.18	−3.06	−0.46	0	−5.34	−1.98	0	0	0.03	0.43	0.2	0	33.27	
6476333‐7	−10.04	−10.04	−2.64	−0.31	0	−5.23	−2	−0.16	0	0.1	0.07	0.14	0	41.38	
6476333‐8	−9.99	−9.99	−3.02	0	0	−5.55	−1.68	−0.06	0	0.02	0.09	0.2	0	40.8	
6476333‐9	−9.72	−9.72	−3.09	0	0	−5.08	−2	−0.08	0	0.27	0.06	0.2	0	29.53	
6476333‐10	−9.68	−9.68	−2.86	0	0	−5.51	−1.85	0	0	0.07	0.33	0.14	0	33.55	
6476333‐11	−9.66	−9.66	−2.6	−0.25	0	−4.93	−2	−0.11	0	0.03	0	0.2	0	42.85	
6476333‐12	−9.55	−9.55	−4.09	−0.26	0	−4.65	−1.66	−0.18	0	1.08	0.08	0.14	0	42.99	
6476333‐13	−9.4	−9.4	−3.02	0	0	−5.33	−2	−0.11	0	0.19	0.67	0.2	0	29.52	
6476333‐14	−9.39	−9.39	−2.46	0	0	−5.95	−2	0	0	0.07	0.81	0.14	0	43.41	
6476333‐15	−9.39	−9.39	−3.04	0	0	−4.59	−2	0	0	0	0.04	0.2	0	40.86	
6476333‐16	−9.32	−9.32	−2.76	0	0	−4.92	−2	0	0	0	0.22	0.14	0	41.13	
6476333‐17	−9.3	−9.3	−2.1	0	0	−5.79	−2	0	0	0.01	0.39	0.2	0	37.02	
6476333‐18	−9.23	−9.23	−3.57	0	0	−4.92	−1.96	0	0	1.03	0	0.2	0	32.88	
6476333‐19	−9.22	−9.22	−2.02	0	0	−5.71	−2	0	0	0	0.3	0.2	0	39.82	
6476333‐20	−9.07	−9.07	−2.69	−0.33	0	−4.77	−1.55	−0.13	0	0.11	0.09	0.2	0	33.53	
6476333‐21	−9.01	−9.01	−2.63	0	0	−5.28	−2	−0.18	0	0.03	0.85	0.2	0	38.14	
6476333‐22	−8.96	−8.96	−3.2	0	0	−3.96	−1.73	−0.21	0	0	0	0.14	0	32.16	
6476333‐23	−8.95	−8.95	−2.23	0	0	−5.13	−2	0	0	0.06	0.22	0.14	0	36.38	
5281718	−8.95	−8.95	−2.71	0	0	−3.97	−1.9	−0.36	−0.2	0	0	0.19	0	31.62	
65056	−8.95	−8.87	−2.85	0	0	−4.4	−1.85	0	−0.03	0.06	0	0.12	0.07	28.22	
6476333‐24	−8.86	−8.86	−2.11	−0.3	0	−4.78	−2	−0.13	0	0	0.27	0.2	0	39.99	
6476333‐25	−8.83	−8.83	−3.22	0	0	−4.35	−2	−0.18	0	0.79	0	0.14	0	38.78	
65056‐2	−8.81	−8.73	−2.95	−0.2	0	−3.75	−2	0	−0.03	0	0	0.12	0.07	32.73	
6476333‐26	−8.55	−8.55	−2.78	0	0	−4.18	−1.75	0	0	0	0.02	0.14	0	35.55	
65056‐3	−8.29	−8.21	−3.03	0	0	−4.36	−2	0	−0.03	0.96	0.05	0.12	0.07	27.97	
5320823	−8.09	−8.06	−2.75	0	0	−3.37	−1.68	0	−0.35	0	0	0.05	0.04	28.41	
6476333‐27	−8.03	−8.03	−1.92	0	0	−5.33	−2	0	0	0	1.09	0.14	0	40.82	
6476333‐28	−7.9	−7.9	−3.09	0	0	−4.34	−1.67	0	0	1	0	0.2	0	42.44	
15689656	−7.85	−7.85	−2.36	0	0	−4.19	−1.39	−0.02	0	0	0	0.11	0	52.64	
5281718‐2	−7.84	−7.84	−2.22	0	0	−3.69	−2	0	−0.2	0	0	0.27	0	32.18	
5281718‐3	−7.82	−7.82	−3.29	0	0	−3.06	−1.88	0	−0.2	0.34	0	0.27	0	33.4	
5281718‐4	−7.82	−7.82	−2.91	−0.25	0	−3.71	−1.44	0	−0.2	0.41	0	0.27	0	27.45	
5281718‐5	−7.81	−7.81	−2.93	0	0	−3.05	−1.91	0	−0.2	0	0	0.27	0	30.91	
5281718‐6	−7.6	−7.6	−3.09	−0.25	0	−2.86	−1.58	0	−0.2	0.11	0	0.27	0	28.38	
15689656‐2	−7.55	−7.55	−2.73	0	0	−3.91	−1.05	−0.02	0	0	0	0.16	0	47.46	
5281718‐7	−7.48	−7.48	−2.73	0	0	−3.18	−1.65	0	−0.2	0.01	0	0.27	0	33.87	
15689656‐3	−7.48	−7.48	−2.63	0	0	−3.88	−1.12	0	0	0	0	0.16	0	50.52	
15689656‐4	−7.41	−7.41	−2.64	0	0	−3.74	−0.91	−0.23	0	0	0	0.11	0	50.38	
5281718‐8	−7.34	−7.34	−2.34	0	0	−3.27	−1.81	0	−0.2	0	0	0.27	0	29.33	
5281718‐9	−7.26	−7.26	−2.91	0	0.65	−2.6	−2.4	0	−0.2	0	0	0.19	0	30.82	
5281718‐10	−7.22	−7.22	−2.11	0	0	−3.47	−1.71	0	−0.2	0	0	0.27	0	31.4	
5281718‐11	−7.2	−7.2	−2.81	0	0	−3.2	−1.28	0	−0.2	0	0	0.27	0	30.11	
5281718‐12	−7.2	−7.2	−2.52	0	0	−3.16	−1.6	0	−0.2	0	0	0.27	0	30.64	
6476333‐29	−7.2	−7.2	−2.99	−0.28	0	−6.31	−2	−0.18	0	4.06	0.36	0.14	0	33.39	
5281718‐13	−7.19	−7.19	−2.5	0	0	−3.13	−1.63	0	−0.2	0	0	0.27	0	30.06	
15689656‐5	−7.18	−7.18	−2.45	0	0	−3.73	−1.13	0	0	0.01	0	0.11	0	49.67	
72281	−7.19	−7.18	−2.72	0	0	−2.74	−1.3	0	−0.49	0	0	0.06	0.02	14.32	
15689656‐6	−7.12	−7.12	−1.9	0	0	−3.74	−1.65	0	0	0	0	0.16	0	49.03	
15689656‐7	−7.04	−7.04	−2.66	0	0	−3.28	−1.35	0	0	0.09	0	0.16	0	51.2	
15689656‐8	−7.03	−7.03	−3.53	0	0	−2.75	−0.93	0	0	0.02	0	0.16	0	53.71	
65056‐4	−7.07	−6.99	−2.65	0	0	−3.04	−1.3	−0.22	−0.03	0	0	0.18	0.07	26.16	
5281718‐14	−6.94	−6.94	−2.75	0	0	−2.32	−1.93	0	−0.2	0.03	0.05	0.19	0	28.86	
15689656‐9	−6.94	−6.94	−1.35	0	0	−4.5	−1.25	0	0	0	0	0.16	0	52.44	
15689656‐10	−6.93	−6.93	−1.71	0	0	−4.48	−0.9	0	0	0.01	0	0.16	0	55.89	
5281718‐15	−6.88	−6.88	−3.15	−0.25	0	−2.7	−0.86	0	−0.2	0.01	0	0.27	0	30.9	
5281718‐16	−6.86	−6.86	−2.46	0	0	−3.1	−1.39	0	−0.2	0.01	0	0.27	0	28.73	
5281718‐17	−6.85	−6.85	−2.69	0	0	−2.07	−2	−0.17	−0.2	0	0	0.27	0	31.23	
5281718‐18	−6.85	−6.85	−2.14	−0.12	0	−2.77	−1.84	0	−0.2	0.03	0	0.19	0	31.55	
5281718‐19	−6.82	−6.82	−2.45	0	0	−3.28	−1.46	0	−0.2	0.3	0	0.27	0	30.26	
5281718‐20	−6.81	−6.81	−2.47	0	0	−2.59	−1.84	0	−0.2	0.02	0	0.27	0	27.99	
15689656‐11	−6.81	−6.81	−3.03	0	0	−2.65	−1.42	0	0	0.14	0	0.16	0	49.69	
15689656‐12	−6.74	−6.74	−3.08	0	0	−3.1	−1.2	0	0	0.48	0	0.16	0	48.97	
5281718‐21	−6.72	−6.72	−2.02	−0.17	0	−2.61	−2	0	−0.2	0	0	0.27	0	32.29	
6476333‐30	−6.7	−6.7	−2.65	0	0	−6.28	−1.97	−0.12	0	4.02	0.08	0.2	0	41.38	
5281718‐22	−6.66	−6.66	−3.04	0	0	−2.23	−1.51	0	−0.2	0	0.05	0.27	0	28.45	
5315263	−6.65	−6.65	−3.13	0	0	−2.2	−1.11	0	−0.25	0	0	0.04	0	37.99	
5281718‐23	−6.64	−6.64	−2.38	0	0	−2.32	−2	−0.02	−0.2	0	0	0.27	0	33.52	
5281718‐24	−6.62	−6.62	−2.39	0	0	−4.05	−1.92	0	−0.2	1.76	0	0.19	0	31.55	
15689656‐13	−6.6	−6.6	−2.18	0	0	−3.54	−1.04	0	0	0	0	0.16	0	49.11	
15689656‐14	−6.58	−6.58	−1.54	0	0	−3.71	−1.45	0	0	0.01	0	0.11	0	54.17	
5281718‐25	−6.54	−6.54	−2.99	−0.25	0	−2.26	−1.23	0	−0.2	0.11	0	0.27	0	29.27	
65056‐5	−8.09	−6.52	−2.75	0	−1.3	−4.35	−0.82	−0.22	−0.03	1.25	0	0.12	1.57	42.3	
65056‐6	−8.33	−6.49	−2.91	0	0	−4.41	−1.06	−0.13	−0.03	0.03	0	0.18	1.84	32.22	
5281718‐26	−6.46	−6.46	−2.89	−0.25	0	−2.38	−1.14	0	−0.2	0.16	0.05	0.19	0	31.71	
5281718‐27	−6.44	−6.44	−3.09	−0.25	0	−2.06	−1.09	0	−0.2	0	0.05	0.19	0	34.84	
86289353	−6.42	−6.42	−3.18	0	0	−1.9	−1.05	0	−0.35	0	0	0.05	0	28.7	
15689656‐15	−6.38	−6.38	−1.48	0	0	−3.32	−1.6	−0.09	0	0	0	0.11	0	54.67	
5281718‐28	−6.38	−6.38	−2.86	0	0	−2.08	−1.51	0	−0.2	0	0	0.27	0	31.73	
65056‐7	−8.14	−6.3	−3.05	0	0	−4.21	−1.92	0	−0.03	0.94	0	0.12	1.84	33.61	
15689656‐16	−6.29	−6.29	−1.91	0	0	−2.8	−1.74	0	0	0	0	0.16	0	51.7	
15689656‐17	−6.26	−6.26	−1.77	0	0	−3.17	−1.2	−0.29	0	0	0.06	0.11	0	52.77	
65056‐8	−7.78	−6.21	−3.13	0	0	−3.62	−1.44	0	−0.03	0.31	0	0.12	1.57	39.76	
5281718‐29	−6.18	−6.18	−2.05	0	0	−2.64	−1.56	0	−0.2	0.08	0	0.19	0	36	
6476333‐31	−6.16	−6.16	−3.16	−0.4	0	−5.02	−2	−0.18	0	4.03	0.44	0.14	0	36.14	
5281718‐30	−6.09	−6.09	−1.5	0	0	−3.66	−0.92	0	−0.2	0	0	0.19	0	35.65	
Note: Values in the columns representing PhobEnHB is 0; while the highest negative vale denotes the best docking score; DocScore <−6.00 are excluded from the table (Table S6). Gscore means, Glide score; Doc score means docking score; PhobEn means hydrophobic enclosure reward; pairHB means paired hydrogen bond; Electro means electrostatic interactions; LowMW means reward for ligands with low molecular weight; Expos penal means penalty for solvent‐exposed ligands; Rot penal means rotatable penalty.

Figure 7A shows the docked ligand pose in the binding cleft of the receptor in surface view, whereas Figure 7B shows all the possible good affinities with the receptor. Figure 7C represents the nonbonded interaction plot between the receptor and ligand. Here we can identify five hydrogen bonds between ARG4, ASP59, ARG282, and SER11, where ARG4 contributed to two hydrogen bond formations. Three pi‐alkyl or alkyl interactions were observed between the ligand and PHE81, ILE61, and TYR7. Besides, one pi–pi stacked, pi‐sigma, and pi‐anion interactions were identified between the ligand and PHE27, TYR7, and GLU 195, respectively.

FIGURE 7 The binding poses of the ligand CID‐6476333 tautomer‐1 with the receptor Protein Data Bank ID: 3N94. (A) The bound ligand in the binding pocket; (B) all the possible good interactions between the ligand and the receptor; (C) two‐dimensional plot of the interactions between the ligand and receptor.

Important structural interactions between the ligand and the receptor were also analyzed. In the beginning, we searched for hydrophobic interaction within the pocket region that aligns with the complementary ligand regions (Figure 8A). The regions of the receptors are shown in corey‐pauling‐koltun (CPK) mode, whereas the interacting ligand regions are in the ball‐and‐stick model. The figure shows a very good interaction pattern within the cleft, and no hydrophobic molecules were observed at the edge of the pocket. An important interaction was observed in the case of the ligand CID‐6476333 tautomer‐1, which are the hydrogen bonds within the hydrophobic cleft. The bonds were formed between PHE24 and PHE27 (Figure 8B). These bonds ensured tight binding between the ligand and the receptor in association with the hydrophobic interactions. 45 We also analyzed the rotatable bonds in the binding pocket where more rotatable bonds refer to lose fit and vice versa due to continuous conformational changes. 46 We found several rotatable bonds of the ligands that should be considered during modifications and optimizations. These bonds are highlighted in Figure 8C. As the cellular environment contains solvents, the hydrophilic regions of the ligand exposed to the solvent can cause ligand detachment and off‐target attachment. We found one hydrophilic atom of the ligand exposed to the solvent (Figure 8D), which is not much of a threat to the complex, but substitution or subtraction can improve the binding pattern.

FIGURE 8 Analysis of the docked complex considering some important factors. (A) The hydrophobic interaction pattern within the pocket region, (B) the hydrogen bond within the hydrophobic interacting region, (C) the rotatable bonds of the ligand within the binding cleft, and (D) the solvent‐exposed hydrophilic region of the ligand from the ligand–receptor complex.

3.10 Effects of AODE on pharmacokinetic and toxicological properties

3.10.1 Target protein identification using network pharmacological analysis

We obtained 488 unique target proteins using SwissTargetPrediction (Table S2). We found that the 19 compounds interacted with target proteins with a probability of >0.1 (Table S1). For example, the interaction of ciwujiatone with 38 compounds is shown in Figure 9A.

FIGURE 9 (A) The interaction of the most active compound ciwujiatone (yellow color) with the 38 target proteins. Figures were generated using Cytoscape, version 3.6.1. (B) The interaction of the top 10 hub genes with active compounds. (C) The top‐enriched GO (Gene Ontology) is significantly associated with target proteins, and yellow represents the hub genes and light green the interacting compounds of AODE.

3.10.2 Effect of AODE on target PPI network construction and identification of hub nodes

To construct the PPI, the target proteins were loaded onto STRING‐DB, version 11.0 (Figure 9B). A total of 475 target proteins were found to be involved in PPI with the following parameters: p < 1.0e−16; number of edges, 7351; average node degree, 30.7; and average local clustering coefficient, 0.444. The PPI network nodes were subsequently examined using three topological parameters: degrees, degrees of centrality, and closeness centrality. Additionally, the rank of the interacted target proteins was identified. Most interactive degrees indicate potential biological functions. The first screening yielded an initial interaction network diagram consisting of 208 hub genes (degrees not less than 25), which are presented in Table S2. Figure 9C shows the leading 10 hub genes. The hub nodes were AKT1 (protein kinase B), CTNNB1 (catenin beta‐1), SRC (proto‐oncogene c‐Src), TNF (tumor necrosis factor), EGFR (epidermal growth factor receptor), HSP90AA1 (heat shock protein 90α), MAPK3 (mitogen‐activated protein kinase 3), STAT3 (signal transducer and activator of transcription 3), CASP3 (caspase protein), and ESR1 (estrogen receptor 1) as core targets.

Moreover, the hub genes were found to interact with many active compounds. For example, estrogen receptor 1 (esr1) interacted with 10 active compounds, including (3R)‐4′‐methoxy‐2′,3′,7‐trihydroxy‐isoflavanone, 5,7,2′,3′,4′‐pentamethoxyflavone, 6‐gingerol, gigantol, hesperetin, licoflavone A, norkurainol, octahydrocurcumin, silandrin, and stilbostemin D (Figure 9C). However, the most active compound was found to be ciwujiatone, which interacted with 38 genes.

3.10.3 GO analysis of interacting target proteins

The GO enrichment analysis of interacting target proteins that interact with the compound was performed using DAVID. We input the 488 target proteins into the DAVID tool and selected the species Homo sapiens. The identified GO are presented in Tables S3–S5. The top 10 BPs, MFs, and CCs are shown in Figure 10.

FIGURE 10 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of hub targets related to liver injury. GO enrichment analysis of top 10 hub targets for biological processes (BP), cellular components (CC), and molecular functions (MF).

3.10.4 KEGG pathways for enrichment analysis of target proteins

GO and KEGG enrichment analyses of hub targets related to liver injury are shown in Figure 10, where the GO enrichment analysis of the top 10 hub targets are shown for BPs, CCs, and MFs. The KEGG pathways, significantly associated with the target proteins, were identified to further elucidate the relationship between target proteins and the pathways (Table S6). These pathways were mostly engaged in signaling pathways for cancer. The top enriched pathways are hsa05200:pathways in cancer, hsa05215:prostate cancer, hsa01521:EGFR tyrosine kinase inhibitor resistance, hsa01522:endocrine resistance, hsa04012:ErbB signaling pathway, hsa04933:AGE‐RAGE, signaling pathway in diabetic complications, hsa04066:HIF‐1 signaling pathway, hsa05161:hepatitis B, hsa04151:PI3K‐Akt signaling pathway, hsa05212:pancreatic cancer, hsa04014:Ras signaling pathway, hsa05223:non‐small‐cell lung cancer, hsa04660:T‐cell receptor signaling pathway, hsa04010:MAPK signaling pathway, hsa04919:thyroid hormone signaling pathway, hsa05230:central carbon metabolism in cancer, hsa05205:proteoglycans in cancer, hsa05207:chemical carcinogenesis‐receptor activation, hsa05214:glioma, and hsa04917:prolactin signaling pathway. The 95 genes are involved in the regulation of the hsa05200:pathways in cancer. These signaling pathways with the involvement of 95 genes are shown in Figure 11.

FIGURE 11 The involvement of 95 genes with hsa05200:pathways in cancer, whereas chemical‐induced cancers are embodied with a number of organs and tissues, including liver.

4 DISCUSSION

Plant‐derived therapeutics have made a significant contribution to prevent liver diseases with the development of natural drugs of higher accessibility, lower cost, patient compliance, perceived viability, and fewer side effects. Furthermore, research indicates that plant‐based drugs can manage and treat liver illness, preventing hepatotoxicity. 47 , 48 Therefore, the study demonstrated how AODE protects against hepatotoxicity in vivo and how medicines were produced using a computer‐aided approach to AODE's bioactive components. Using qualitative phytochemical screening and LC‐qTOF‐MS, the phytochemical profiling for this study identified a variety of phytochemicals, phenols, flavonoids, steroids, glycosides, cardiac glycosides, and tannins. The results demonstrate that by causing antioxidative effects to stop ROS or cellular damage, AODE can reduce the risk of hepatotoxicity. Recent studies showed that numerous bioactive metabolites, like tribulusamides A and B, significantly prevented hepatocyte death induced by d‐galactosamine/TNF‐α and functioned as antioxidants to prevent cellular damage by lowering oxidative stress. 49

Toxicity studies of AODE showed that the samples exhibited no signs of toxicity, morbidity, or mortality after applied dosages. The well‐known analgesic and antipyretic drug PCM is safe when used as part of a dosing regimen. On the contrary, both people and animals may suffer liver damage from extensive PCM use. 50 The extremely reactive and dangerous metabolite N‐acetyl‐p‐benzoquinone imine (NAPQI), which is a result of PCM bioactivation, is formed by hepatic cytochrome. NAPQI can cause a variety of biological reactions, such as apoptosis, liver damage, interaction with cellular macromolecules, GSH depletion, and liver damage. 51 Numerous cellular enzymes that can affect hepatocyte transport processes are released into the serum because of toxicity. Because the liver is a vital metabolic organ in the living body and is affected by several substances and toxicants, hepatotoxins frequently harm the liver. 50

Hepatotoxicity induced by PCM is the ultimate outcome of liver damage, which can also manifest through abnormalities in various biochemical markers of liver enzymes and metabolites. PCM interacts with biological proteins to produce mitochondrial malfunction, which results in widespread liver cell death and necrosis. 52 As a result of excessive PCM use, several biomarker values in the blood change. Different serum profiles, including increased ACP, AST, ALP, ALT, GGT, LDH, and TB levels, as well as decreased TP level in response to excessive PCM exposure, were shown by the trial. On the contrary, treatment with AODE at different doses decreased the anomalies of the blood serum marker, enabling the management of the hepatic cellular membrane's integrity.

Hepatoprotective action has been greatly aided by a variety of exogenous and endogenous antioxidant enzymes. 53 Overproduction of ROS, which causes numerous cellular changes and interacts with a wide range of molecules, including proteins, DNA, and lipids, leading to the decrease in the antioxidative enzymes SOD, GSH, and CAT, is the primary cause of PCM‐stimulated hepatotoxicity. The results of the study show that AODE treatment lowers MDA level and shows a good improvement in SOD, GSH, and CAT. The mRNA expression levels of five antioxidant genes (CAT, SOD2, β‐actin, PON1, and PFK‐1) further corroborate our earlier findings from several investigations. According to our research, the disruption of the antioxidant defense mechanism is the important stage in PCM‐induced damage. All five antioxidant genes have been shown to be significantly downregulated in the PCM‐induced group in a dose‐dependent manner. On the contrary, AODE pretreated groups discovered high levels of overexpression of all antioxidant genes using the reference standard medication silymarin. This discovery was confirmed by earlier research conducted by other scientific organizations. 54 , 55

We also attempted to determine whether the plant extract could protect the liver, reduce oxidative stress, or stop fibrosis in rats receiving PCM. At maximum AODE administration, we found that liver tissue exhibited a regular lobular pattern with a minor degree of necrosis and lymphocytic infiltration, essentially identical to the NC and almost similar to the reference standard medication silymarin. On the contrary, the intermediate and lowest doses produced mild‐to‐moderate hepatic problems. Histological data show that some regions of the liver underwent gradual changes as a result of PCM exposure. Numerous clinical conditions, including inflammation, necrosis, sinusoidal dilation, ballooning degeneration, apoptosis, bleeding, and other histological symptoms, have been related to PCM as the pathogenesis, which is consistent with earlier findings. 56 The results of this investigation indicate that AODE reduces hepatotoxicity in a dose‐dependent manner. The primary bioactive elements were categorized in this study using network pharmacology, a highly effective and economical method, to obtain a full evaluation of the mechanisms of AODE on the hepatoprotective effects of PCM‐induced liver injury. It is possible to view the 10 target genes AKT1, CTNNB1, SRC, TNF, EGFR, HSP90AA1, MAPK3, STAT3, CASP3, and ESR1 as important elements in interactions with certain AODE drugs. Most of the requirements are met by the Akt gene activity in an unhealthy liver. Although cell survival is the primary function of Akt, studies have shown that Akt also plays significant roles in the uptake of glucose, production of glycogen, progression of cell cycle, and lipid metabolism. 57 With varying rates depending on the etiology, mutations in the CTNNB1 genes are thought to be the primary drivers of the development of hepatocellular carcinoma. 58 Casp3(−/−) animals on a methionine choline–deficient diet had lower hepatic collagen deposition due to reduced profibrogenic gene transcript expression. Apoptosis and inflammatory signaling cytokines reduced significantly due to these alterations. 59 SRC maintained the framework of cells, although it is known that it may disrupt the fundamental cellular functions of liver cancer, 60 and Srebf1, Srebf2, Fasn, Acc1, and Ppara were among the genes induced by EGFR both in vitro and in vivo. On a molecular level, EGFR enhances TGF‐1/Smad signaling and makes it difficult for TGF‐1/EGFR to stop hepatocyte genes from producing fat. 61 In humans, the CYP19A1 gene encodes aromatase. Lack of aromatase impairs adiposity, lipid profiles, liver function, and glucose metabolism, all of which contribute to estrogen deprivation. 62 This interaction with the target molecules may mitigate liver damage and provide protection. As part of the cellular response to damage, MAPKs regulate a variety of activities, including development, inflammation, metabolism, and apoptosis. MAPK inactivation facilitates DNA replication during liver regeneration, which gently portends a considerable influence on the hepatic condition recovery mechanism after PCM‐induced damage. 63 , 64 Additionally, MAPK3, called extracellular signal‐regulated kinase 1, is an important ERK/MAPK pathway cell signaling molecule that is documented as a pivotal gene of the hub gene components. MAPK3 usually mediates the transmission of signals from a cell's exterior to its interior. 65 The overexpression and/or hyperactivity of MAPK3 has been linked to the initiation, development, and drug resistance in various carcinomas, including liver cancers. 66 Therefore, the effect of AODE component, especially the most active compound, ciwujiatone, could be one of the potential inhibitors of MAPK3 in chemical‐induced liver injury.

It has been demonstrated that estrogen stimulates hepatocyte proliferation, mostly through ESR1. 67 Ten hub genes (degree not less than 10) were present in the network, indicating shared regulatory connections between the targets and components of AODE compounds. Further docking studies confirmed the inhibitory potentials of these compounds. Though all of them did not exhibit maximized efficacy in binding with the receptor, optimization of these lead compounds can improve the affinity significantly. The ligand we discussed had 32 tautomers (the condition was bound to that limit), which were due to its rotatable bonds. This compound possesses about 11 rotatable bonds and ultimately results in a higher number of structural conformers. If we can limit the number of rotatable axes, it will cause fewer conformational changes, with rigid binding consisting of higher affinity. The complex also exhibited strong hydrophobic interaction along with stable hydrogen bonds within the hydrophobic region of the receptor pocket, referring to the higher stability of the docked complex. As a result of this special type of interaction, detachment will be nearly impossible as it will require overcoming the van der Waals attraction force but also the strong hydrogen bonds. Another important discovery of this interaction was the negligible solvent–exposed hydrophilic surface of the ligand, which has almost no effect on the cellular environment against docking. But the removal of the oxygen atom or substitution with a hydrophobic group like methyl (–CH3) or benzene (C6H6) can increase the binding affinity of the complex as the nearest pocket region of the hydrophilic atom is rich in hydrophobic amino acids.

The target protein's GO and pathway enrichment analyses were performed. In addition to confirming its involvement in oxidative stress–related pathways, GO analysis revealed that the top few enriched biological systems were responses to oxygen‐containing compounds, lipid metabolic processes, transition metal ion binding, ligand‐activated transcription factor activity, and positive regulation of map kinase activity. These results corroborate those of a molecular investigation showing that some chemicals significantly increased the expression of an antioxidative enzyme. KEGG enrichment analysis enabled us to identify the pathways involved in the drug's hepatoprotective properties against PCM‐induced liver damage. The liver is able to regenerate itself after injury because of a dynamic cellular network that orchestrates proliferative and hepatoprotective signaling cascades. Because the chemicals are part of the calcium signaling pathway, hepatocyte turnover is enhanced, and cytosolic Ca2+ levels are increased. 68 These chemicals also interacted with cancer‐related pathways, providing a promising avenue for further research into the possibility of assessing their anticancer potential. The findings of PPI networks and GO and KEGG enrichment analyses reveal that compounds against PCM‐induced hepatotoxicity may be involved in processes such as liver regeneration, oxidative stress control, fatty acid management, and anti‐inflammatory action.

5 CONCLUSIONS

The study results confirmed AODE's ability to reverse PCM‐induced liver injury. The biochemical markers were changed, antioxidant enzymes were protected, and the levels of CAT, SOD, β‐actin, PON1, and PFK‐1 increased, all the while PCM was downregulated because of AODE treatment. In addition, the top 12 molecules interacting with receptors were identified using computer‐assisted drug design; these compounds have drug‐like features, are nontoxic, and may even have hepatoprotective effects. Finally, genes with antioxidant and tissue regeneration functions, such as AKT1, CTNNB1, SRC, TNF, EGFR, HSP90AA1, MAPK3, STAT3, CASP3, and ESR1, were identified using network pharmacology analysis as being important in protecting against and recovering from liver damage or toxicity caused by PCM in the model.

AUTHOR CONTRIBUTIONS

Md. Atiar Rahman: conceptualization, resources, supervision, project administration, funding acquisition, writing—review and editing; A. M. Abu Ahmed: investigation, methodology, data curation, formal analysis, writing of the original draft; Farjana Sharmen, A. S. M. Ali Reza, Md. Shahidul Islam, and Md. Mamunur Rashid: investigation, resources, data curation; Md. Khalid Juhani Rafi and Tanvir Ahmed Siddiqui: investigation, validation, writing—review and editing, visualization; Md. Muzahid Ahmed Ezaj: software, formal analysis, writing of the original draft; Srabonti Saha: investigation, validation, resources; Md. Nazim Uddin: formal analysis, investigation; Walla Alelwani: resources, review and editing. All the authors have read the manuscript and agreed to submit for publication.

FUNDING INFORMATION

None.

CONFLICT OF INTEREST STATEMENT

The authors declare that they do not have any conflicts of interest.

ETHICS STATEMENT

The experimental animals were handled and maintained according to the recommended protocol, established under the ARRIVE guidelines, of the Institutional Animal Ethics Committee, Faculty of Biological Science, University of Chittagong, with a reference number of (AERB‐FBSCU/2023‐03).

Supporting information

Tables S1–S6.

ACKNOWLEDGMENTS

The authors thank Dr. Mohammad Musharof Hossain and Dr. Shaikh Boktear Uddin, both professors of botany at the University of Chittagong, Chittagong, Bangladesh, for their help in positively identifying the sample.

DATA AVAILABILITY STATEMENT

Data will be available upon request.
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