The Structural, Biological, and In-Silico Profiling of Novel Capryloyl Tetra-Glucoside and Aliphatic Ester Constituents from the Abutilon indicum Offers New Perspectives on the Treatment of Pain and Inflammation

Abutilon indicum L. (Malvaceae), more often referred to as Peeli booti, Kanghi, and Kakhi, is a perennial shrub found in many countries of Asia. Traditionally, this plant is used as a diuretic to treat inflammation, discomfort, urethral infections, and gout. Inflammation and pain are key topics of interest for researchers throughout the globe, since they are linked to almost every illness that could affect humans or animals. The present study was conducted to isolate the phytoconstituents from the methanolic extract of Abutilon indicum collected from the Bihar state Koshi river belt in India, and to evaluate the isolated phytoconstituents’ ability to reduce nociception and inflammation. Furthermore, molecular docking was performed to investigate the molecular interaction profile, with possible therapeutic targets for anti-inflammatory medicines. A. indicum methanolic extract yielded two novel phytocompounds identified as 5′-hydroxyhexyl n-hexadecanoate (AB-01) and n-octanoyl-β-D-glucopyranosyl-(2′-1′′)-β-D-glucopyranosyl-(2′′-1′′′)-β-D-glucopyranosyl-(2′′′-1′′′′)-β-D-glucopyranoside (AB-05), together with three previously recognized phytocompounds such as ester glucoside. All isolated molecules were tested for the efficacy of analgesic and anti-inflammatory characteristics at doses of 5 and 10 mg/kg body weight. The isolated compound’s molecular interaction profile with anti-inflammatory drug targets cyclooxygenase-2 and tumor necrosis factor-alpha possessed high potential energy in molecular docking. These findings may aid in developing anti-inflammatory and analgesic drugs from A. indicum.


Introduction
Abutilon indicum L. (Malvaceae), known locally as Peeli booti, Kanghi and Kakhi, is a perennial shrub found in many countries of Asia [1]. Ethnobotanical surveys have found that nearly all plant parts are healthy and have been used for centuries to treat various diseases. Roots and leaves are used as diuretics and demulcents to treat urethritis and chest dish and continuously cooked in a water bath by progressively adding methanol (100 mL) in small parts with constant stirring until the desired consistency was achieved. A weighed quantity of silica gel for column chromatography (Merck, Mumbai, India) was gently added with continuous mixing with a steel spatula until the entire methanolic solution of plant extract was adsorbed onto silica gel particles. It was air-dried and rubbed between hands to break up the larger lumps before passing through a sieve (No. 8), to achieve uniform particle size.
A 16-mm-diameter 5-foot-tall column was obtained, cleansed, and dried. Nonabsorbent cotton wool was used to plug the lower end of the column. The column was fastened and placed on a stand in a vertical position. Petroleum ether (b.p. 60-80 • C) was then half-filled into the column. The dried plant extract slurry was put into the column and then eluted sequentially with different solvents of increase polarity. Silica gel (for the column, 60-120 mesh, Merck, Mumbai, India) was then poured in small parts and allowed to settle down. The column was developed and eluted with a series of different solvents in various combinations, including petroleum ether, petroleum ether: chloroform (9:10, 3:1, 1:1, 1:3 v/v), chloroform, chloroform: methanol (99:1, 98:2, 97:3, 24:1, 19:1, 9:1, 3:1, and 1:1, 1:3 v/v), chloroform. Different ratios were taken and tested using TLC to assess the homogeneity (Silica gel G precoated TLC plates, Merck, Mumbai, India). Fractions with identical Rf values were combined and crystallized with solvents. Their spots were visualized by exposing them to iodine vapors, UV radiations (254 and 366 nm) and spraying with 0.1% anisaldehyde sulfuric acid as spraying reagents. The separated phytochemicals were then recrystallized to obtain pure compounds. The compound obtained from the column was crystalline pure. It showed clear Rf, melting point, and UV lambda max values. The purity of the compound was also checked by the hydrolysis of compounds and the Rf value of individual components.

General Experimental Procedures
Methanol (Merck, Mumbai, India) was used as a solvent in the Shimadzu-120 double beam spectrophotometer to determine Ultraviolet spectra. Shimadzu FTIR-8400 was used to detect and document IR spectra in KBr pellets. Scanning of 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra was carried out using the Bruker spectrospin NMR apparatus exploiting TMS as the internal standard. EIMS at 70 eV with a Jeol D-300 device (Jeol, USA) was used for scanning, and silica gel (60-120 mesh, Merck, Mumbai, India) was used for column chromatography. For thin-layer chromatography, silica gel G-coated TLC plates manufactured by Merck in Mumbai, India, were used. For thin-layer chromatography, silica gel G-coated TLC plates (Merck, Mumbai, India) were employed. Based on the dried plant material acquired for extraction (4.8 kg), an estimate of the % yields of the isolated compounds were determined.

Animals
Adult Swiss albino mice of both sexes, weighing between 25 and 30 g, were used for the analgesic activity study, while healthy Wistar albino rats of both sexes, weighing around 140 and 160 g, were used for the anti-inflammatory activity study. They were kept in clean, sterilized polypropylene cages at ambient temperature (21 ± 2 • C) and under a dark/light control for 12 h. Additionally, they were fed commercial pellets and had access to water ad libitum. Mice were randomly assigned to groups and kept in isolation for a week to allow for sociability and acclimation to their new surroundings and caretakers before the experiments. The research was approved (Phar-02/2018) and conducted in accordance with the recommendations of the Institutional Ethical Committee of the Faculty of Pharmacy at Misurata University in Misurata, Libya.

Safety Profile Study
An acute toxicity study was conceded for the assurance of LD 50 by carrying out the plan (Annexure 2d) of CPCSEA, OECD guideline No. 423. Swiss albino mice of each sex were divided into seven groups, with six animals in each group. Isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 from a methanolic extract of A. indicum at various dose levels of 50, 100, and 150 mg/kg were administered orally as a single dose to mice. The animals were observed periodically for indications of toxicity and death for 24 h, and afterwards consistently for 14 days [26].

Administration of Drugs
In all the experimental animals, the isolated chemicals AB-01, AB-02, AB-03, AB-04, and AB-05 were given at 05 and 10 mg/kg dosages. In the hot plate approach, Indomethacin 10 mg/kg was used as a typical anti-inflammatory medicine, and Tramadol 0.1 mL (40 mg/kg. s.c.) was employed as a pain-inhibiting agent. In mice with acetic acid-induced writhing, 5 mg/kg of diclofenac was used as a painkiller. To obtain the necessary dosage on a bodyweight basis (mg/kg) of the animal, all the test and standard drugs were mixed into an emulsion using 0.3 percent carboxy methyl cellulose (CMC). This emulsion was then delivered orally using a safety needle with a ball-finished tip [27].

Carrageenan-Induced Rat Hind Paw Edema
Acute inflammation was incited by the injection of 0.1 mL of 1% freshly prepared suspension of carrageenan in normal saline into the sub-plantar region of the right hind paw of all groups of animals. At 1, 2, 3, 4, and 5 h intervals, the volume of the injected paws was measured using a plethysmometer. The animals were premedicated with the vehicle (0.3% CMC p.o.), isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 at doses 05 and 10 mg/kg, and the standard drug indomethacin (10 mg/kg) 1 h before the carrageenan challenge [28]. The percentage inhibition of inflammation was intended to be consistent with the following formula: % inhibition = 100 (1 − Vt/Vc), where 'Vc' stands for the inflammation volume in the control and 'Vt' for the inflammation volume in the group treated with tested drugs.

Analgesic Activity
The isolated compounds AB-01, AB-02, AB-03, AB-04, and AB-05 were tested for their analgesic effectiveness using a chemical approach (an acetic acid-induced writhing response) and a thermal approach method (hot plate reaction time).

Hot Plate Test
The pain-relieving (analgesic) test was carried out with an Eddy's hot plate, maintained at a temperature of 55 ± 1 • C. The basal response time of all mice towards thermal heat was verified first. They were then treated with the vehicle (0.3% CMC p.o.) along with the isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 at doses of 05 and 10 mg/kg and the standard medication Tramadol 0.025 mL (10 mg/kg. s.c). Following an hour of testing and standard medication dosing, the mice in all groups were placed individually on the hot plate, which was maintained at 55 • C. The snapshot of time required in seconds for paw licking or bouncing was noted as reaction time. A remove phase of 30 s was maintained to avoid harm to the paw. The pain inhibition percentage (PIP) was determined by the accompanying equation [29]: where T 1 is post-drug latency, and T 0 is pre-drug latency time.
Acetic Acid-Induced Writhing Test The animals were pre-medicated with the vehicle (0.3% CMC p.o.), AB-01, AB-02, AB-03, AB-04 and AB-05 at doses 05 and 10 mg/kg, and the standard medication diclofenac (5 mg/kg). Acetic acid (1% v/v) in doses of 1 mL/kg body weight was injected intraperitoneally in all the groups of animals 1 h after dosing of the test and standard drugs. Writhing was recorded by counting the number of writhes following the injection of acetic

ADMET Analysis
Chemical Scheme of all the test compounds (AB 01 to AB 05) were drawn up using Chemdraw Ultra 12.0 and converted to mol files. These files were then imported to Maestro 9.0 workspace and subjected to ligand preparation. Once the ligands had been prepared, these were subjected to the Qikprop tool for absorption, distribution, metabolism, excretion and toxicology (ADMET) profiling [30,31].
2.6. Molecular Docking 2.6.1. Ligand Preparation Molecular docking methods are frequently employed to compute the binding affinities of a range of ligands [30,[32][33][34]. The structure of the isolated molecules was drawn in a chem sketch and shown to possess high energy. Therefore, an energy minimization approach was applied to optimize the molecule structures. Energy minimization of the ligands was carried out using the 'Minimize Ligands' module from Biovia Discovery Studio, employing the CHARMm forcefield and Smart Minimizer algorithm for 2000 steps. The smart Minimizer method made the original structure different from the minimal structure. All the minimized ligands were converted to pdbqt before the docking procedure [33,35].

Protein Preparation
The TNF-alpha receptor protein (PDB ID: 2AZ5_A) was retrieved from the RSCB protein data bank (http://www.rcsb.org/) (accessed on 11 April 2022). Each protein was cleaned of water molecules and co-crystal ligands. To produce pdbqt files, the protein was assigned polar hydrogens, Kollman charges, solvation parameters, and fragmental volumes using the Graphical User Interface tool AutoDock Tools (ADT) Version 4.2. To generate a grid around the protein, ADT was used to create a grid around the binding pocket. A grid map was created using AutoGrid and a grid box. The grid dimensions were set at −19.163, 74.452, and 33.837 in x, y, and z coordinates in the binding pocket with a grid spacing of 0.375. The grid size was set to 78, 60, and 72 in x, y, and z points in the binding pocket. Using the grid box properties specified in the configuration file, AutoDock Vina was used to dock proteins and ligands. Throughout the docking process, proteins were labelled rigid, whereas ligands were labeled flexible. The findings with a positional root-mean-square deviation (RMSD) less than 1.0 were clustered together and represented by the binding free energy result with the lowest value. The ligand conformation with the lowest binding energy or affinity was selected and fitted with the receptor structure for intra-molecular interaction studies [11,36]. The 'View Interaction' protocol from the Discovery Studio (DS) [Dassault Systems, BIOVIA Corp., San Diego, CA, USA, v 20.1] application was used to conduct the interaction analysis in this study.

Statistical Analysis
Graph Pad Prism V2.01 was used to carry out a one-way variance analysis, followed by Dunnett's post-hoc test (GraphPad Software, Inc., San Diego, CA, USA). Experiments were performed in triplicate and repeated three times. The data were expressed as the mean ± standard error of the mean, and p < 0.05 and <0.01 were considered statistically significant.

Compound (AB-01)-5 Hydoxyl Palmitate
A colorless crystalline mass of AB-01 was obtained when the column was eluted with a mixture of chloroform and methanol in the proportion of 19:1. This was recrystallized with chloroform: methanol in a ratio of 1:1 that yielded 95 mg mass of yield 0.12%. The R f value 0.39 was received for the CHCL 3 -MeOH in a ratio of 19  Hydrolysis of AB-02: Compound 2 (15 mg) was dissolved in ethanol (5 mL). Concentrated hydrochloric acid (2 mL) was then added, and the reaction mixture was heated for 1 h in a steam bath. Following this, it was chilled and extracted using petroleum ether, equivalent to a reference capric acid sample, on a TLC plate developed with petroleum ether to separate the capric acid. After separating the fatty acids using n-butanol-acetic acid water and a reference sugar sample, the residue was concentrated and chromatographed on a silica gel TLC plate (4:1:5, top layer). The sugar was revealed to be D-glucose, according to R F . 0.12.

Compound (AB-03)-Capryl Diglucoside
Compound AB-03 was eluted from the column with a mixture of chloroform and methanol (9:1), resulting in a colorless mass. It was then recrystallised with methanol, which eventually yielded 118 mg (0.16% yield). The R f 0.51 (CHCl 3 -MeOH, 9:1) and m.p. 120-122 • C. The UV λ max (MeOH) was noted at 270 nm. Using KBr-IR ν max was recorded: Hydrolysis of AB-03: Hydrochloric acid (conc.) was added to the reaction mixture with Compound 3 (10 mg), and the reaction took place for one hour in a hot steam bath. Caprylic acid was isolated by using petroleum ether as a solvent. To chromatograph the fatty acid residue over the developing solvent system of n-butanol, acetic acid, and water (4:1:5), silica gel TLC plates with a standard sample of sugars were used. D-glucose, R F 0.12, was identified as the sugar.

Compound (AB-04)-Palmityl Diglucoside
Crystals of AB-04, recrystallized from chloroform: methanol (l:1), were produced by further eluting the column with chloroform: methanol solvents (9:1). The yield was found to be 206 mg in terms of % yield 0. 28. The R f 0.72 (CHCl 3 -MeOH, 9:1) and m.p. were reported to be 105-110 • C. UV λ max (MeOH) was shown at 217 nm. IR ν max ( Hydrolysis of AB-04: The reaction mixture containing Compound 4 (10 mg) was treated with ethanol (5 mL) and concentrated HCI (2 mL) for one hour in a hot steam bath. Palmitic acid was separated from the reaction mixture using chloroform extraction, and the co-TLC findings were then compared to these. After separating the fatty acid, it was concentrated and chromatographed on silica gel TLC. A reference sample of the sugar utilizing the top layer of n-BuOH-AcOH-H2O, 4:1:5 was used. The fatty acid was then separated. D-glucose was identified as the sugar (R f 0.12) and was separated.

Compound (AB-05)-Capryloyl Tetraglucoside
Column elution with methanol: chloroform in a ratio of (1:9) gave a crystalline mass of a light brown color AB-05, which was recrystallised again from methanol that yielded a 145 mg mass of 0.23% yield. The R f 0.63 (CHCl 3 -MeOH, 9:1) and m.p. was found to be 125-126 Hydrolysis of AB-05: A steam bath was used to heat a combination of 15 mg of Compound 5 for 1 h, using conc. HCL 2 mL and ethanol 5 mL. Petroleum ether was employed to extract capric acid from the reaction mixture, and co-TLC results were equivalent. The reaction mixture was concentrated, and the standard sugar was chromatographed on silica gel TLC with n-butanol-AcOH-H 2 O in a ratio of 4:1:5 as the developing solvent system. D-glucose, R F 0.12, was identified as the sugar.

Safety Profile Study
It was seen that the dosing of isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 up to 150 mg/kg orally to the mice did not incite drug-related harmfulness (toxicity) and mortality. The results revealed the mice endured the drug well and showed normal behavior up to 150 mg/kg orally. All animals were vigilant, with normal spruce, touch, and pain response, and there was no indication of accommodation or vocalization.

Carrageenan-Induced Rat Paw Edema
The anti-inflammatory effect of isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 at doses of 5 & 10 mg/kg and Indomethacin at a dose of 10 mg/kg on carrageenandisturbed rat paw is displayed in Figure 1. The anti-inflammatory effects of all tested drugs were recorded from 60 min after the carrageenan challenge. Paw edema in rats reached its highest at 4 h following the carrageenan challenge, and animals treated with Indomethacin showed a significant (p < 0.05) decrease in paw volume of rats from 2 h. Treatment with Indomethacin and AB-01, AB-02, AB-03, AB-04 and AB-05 at 10 mg/kg showed a significant reduction (p < 0.05) of paw volume in rats incited by carrageenan. The impact of AB-01 and AB-05 at a portion of 10 mg/kg was favored in lessening the swollen paw volume, rather than a higher portion of Indomethacin (10 mg/kg).

Hydrolysis of A B-05:
A steam bath was used to heat a combination of 15 mg of Compound 5 for 1 h, using conc. HCL 2 mL and ethanol 5 mL. Petroleum ether was employed to extract capric acid from the reaction mixture, and co-TLC results were equivalent. The reaction mixture was concentrated, and the standard sugar was chromatographed on silica gel TLC with n-butanol-AcOH-H2O in a ratio of 4:1:5 as the developing solvent system. D-glucose, RF 0.12, was identified as the sugar.

Safety Profile Study
It was seen that the dosing of isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 up to 150 mg/kg orally to the mice did not incite drug-related harmfulness (toxicity) and mortality. The results revealed the mice endured the drug well and showed normal behavior up to 150 mg/kg orally. All animals were vigilant, with normal spruce, touch, and pain response, and there was no indication of accommodation or vocalization.

Carrageenan-Induced Rat Paw Edema
The anti-inflammatory effect of isolated compounds AB-01, AB-02, AB-03, AB-04 and AB-05 at doses of 5 & 10 mg/kg and Indomethacin at a dose of 10 mg/kg on carrageenandisturbed rat paw is displayed in Figure 1. The anti-inflammatory effects of all tested drugs were recorded from 60 min after the carrageenan challenge. Paw edema in rats reached its highest at 4 h following the carrageenan challenge, and animals treated with Indomethacin showed a significant (p < 0.05) decrease in paw volume of rats from 2 h. Treatment with Indomethacin and AB-01, AB-02, AB-03, AB-04 and AB-05 at 10 mg/kg showed a significant reduction (p < 0.05) of paw volume in rats incited by carrageenan. The impact of AB-01 and AB-05 at a portion of 10 mg/kg was favored in lessening the swollen paw volume, rather than a higher portion of Indomethacin (10 mg/kg).

Acetic Acid-Induced Writhing Methods
The acetic acid-induced writhing assay was used to evaluate the analgesic efficacy of isolated compounds AB-01 to AB-05 at dosages of 5 and 10 mg/kg, and Diclofenac

ADMET Analysis
ADMET analysis showed that compounds AB 01, AB 02 and AB 03 had a molecular weight below 500; they might be considered drug candidates. In contrast, compounds AB 04 and AB 05, having a molecular weight of more than 500, are poor candidates for drug development. The number of rotational bonds in all the compounds appeared to be more than the acceptable range. With the exception of compound AB 01, all compounds had Figure 3. Effect of isolated compounds AB-01 to AB-05 and Diclofenac (Standard) on acetic acidinduced writhing in mice. Each value is presented as Mean ± S.E.M. One-way ANOVA followed by Dunnett's test, p: * p < 0.05, ** p < 0.01 and *** p < 0.001, compared with respective control group.

ADMET Analysis
ADMET analysis showed that compounds AB 01, AB 02 and AB 03 had a molecular weight below 500; they might be considered drug candidates. In contrast, compounds AB 04 and AB 05, having a molecular weight of more than 500, are poor candidates for drug development. The number of rotational bonds in all the compounds appeared to be more than the acceptable range. With the exception of compound AB 01, all compounds had more than the required hydrogen bond donors, while with the exception of compound AB 05, the remaining compounds had hydrogen bond acceptors within the specified range. With the exception of compound AB 02, all the compounds had partition coefficients within the range. The ADMET analysis result is included in Table 1.

Molecular Docking
The isolated constituents Ab-01 to AB-05 were docked with receptors known to be related to inflammatory pathways. These receptors included cyclooxygenase-2 (PDB id: 3LN1) and tumor necrosis factor-alpha (PDB id: 2AZ5). The docking methodology developed in AutoDock Vina was validated by re-docking all the co-crystallized ligands with their corresponding proteins. In addition, the docked poses of each ligand were compared with their crystal conformations, which exhibited a root-mean-square deviation (RMSD) of 2.0 A • , and validated the dependability of the docking approach used [30,32,33]

Molecular Docking
The isolated constituents Ab-01 to AB-05 were docked with receptors known to be related to inflammatory pathways. These receptors included cyclooxygenase-2 (PDB id: 3LN1) and tumor necrosis factor-alpha (PDB id: 2AZ5). The docking methodology developed in AutoDock Vina was validated by re-docking all the co-crystallized ligands with their corresponding proteins. In addition, the docked poses of each ligand were compared with their crystal conformations, which exhibited a root-mean-square deviation (RMSD) of 2.0 A°, and validated the dependability of the docking approach used [30,32,33]

Discussion
A. indicum is an extensively utilized and accepted traditional medicinal plant. Early findings on the leaf extracts suggested antibacterial, hepatoprotective and larvicidal capabilities, and anti-inflammatory activity [11,[37][38][39]. The study was conducted on a plant native to the area where the study took place, and which is also part of the local diet. Consequently, the present research focused on isolating and characterizing two new phytocompounds and three well-known aliphatic ester glucoside compounds that may have chemotaxonomic significance. The isolated phytoconstituents were screened for possible anti-inflammatory and analgesic therapeutic potential. Further molecular docking was used to clearly understand the analgesic and anti-inflammatory properties of A. indicum. Docking is an essential strategy for developing computerized medications for particular illnesses. Additionally, the online prediction tool ADME analysis was used to determine the pharmacokinetics, drug-likeness, and physicochemical properties of all isolated phytoconstituents. The structural profiling of isolated phytoconstituents from A. indicum methanolic extract yielded two novel phytocompounds, identifying 5 -hydroxyhexyl n-hexadecanoate as AB-01 and n-octanoyl-β-D-glucopyranosyl-(2 -1 )-β-D-glucopyranoside as AB-05. The three previously recognized phytocompounds were also isolated as ester glucoside. Because the exact purity of the isolated compounds was not determined, it is possible that a small fraction of molecules contributed to the observed bioactivities, which might be recognized as a limitation of this study.
Compound AB-01, Eluants from chloroform: methanol (19:1), a colorless fatty acid ester called AB-01, was produced. Its IR spectra ( Figure S1) showed absorption bands for the hydroxyl group (3410 cm −1 ) and the ester function (1722 cm −1 ). The mass spectrum ( Figure S2) at m/z 356, displayed a molecular ion peak, identical to a fatty acid ester's chemical formula, C 22    From chloroform: methanol (19:1) eluants, the crystalline mass of chemical AB-02 capryloyl diglucoside was synthesized. It demonstrated IR absorption bands ( Figure S5) for hydroxyl groups (3450, 3389, 3265 cm −1 ), ester function (1721 cm −1 ), and aliphatic chain (779 cm −1 ) and produced a positive Fehling solution for glucoside. The molecular ion peak of chemical AB-02 ( Figure S6) was identified at m/z 468 based on mass and 13 C NMR spectra, which is compatible with the molecular formula of acyl diglycoside, C 20 H 36 O 12. The ion peaks arising at m/z 127 [CH 3 (CH 2 ) 6 CO] + and 143 [CH 3 (CH 2 ) 6 COO] + indicated that aglycone capric acid was esterified with the diglycosidic glycone unit. The ion fragments generating at m/z 163 [C 6 H 11 O 5 ] + and 326 [M-143, C 12 H 21 O 10 ] suggested that the aglycone unit was connected to the C12 sugar unit (Figure 7). Compound 2 1 H NMR spectra ( Figure S7) revealed two one-proton doublets at δ 4.04 (J = 7.2 Hz) and 3.99 (J = 7.1 Hz), which were attributed to the anomeric protons 1 + -1 and H-1 , respectively. Between δ 3.83-3.10, the other sugar protons made an appearance. Methylene H2-2 near the ester function was found to have a two-proton triplet at around δ 2.56 (J = 7.2 Hz). The acyl unit's methylene protons were responsible for a broad singlet at δ 1.29 and a two-proton multiplet at δ 1.39 (8H). The terminal C-8 primary methyl protons were attributed to a three-proton triplet at about δ 0.88 (J = 6.3 Hz). Compound 2's 13 C NMR spectra ( Figure S8) showed signals for methyl carbon at 14.21δ 14.21(C-8), ester carbon at δ 172.06 (C-1), and anomeric carbons at δ 103.29 (C-1 ) and 99.33 (C-1 ), as well as additional sugar carbons between δ 34.82 and 22.63. The saturated nature of the molecule was corroborated by the lack of any signal in the 1H NMR spectrum above δ 4.04 and the 13C NMR spectrum between δ 172.06 and 130.29. The second sugar unit may have been located at C-2 , as suggested by the presence of C-2 in the deshielded area at δ 83.41 in the 13 C NMR spectra. Capric acid D-glucose was produced via the compound AB-02's acid hydrolysis (TLC comparable). The structure of component AB-02 was determined as n-octanoyl-β-D-glucopyranosyl-2 -β-D-glucopyranoside (Scheme 2) based on these proofs.  Chloroform: methanol yielded capryl diglucoside, molecule AB-03, as a colorless crystalline mass (9:1). It accepted the glycoside Fehling solution test and displayed IR absorption bands for hydroxyl groups (3425, 3389, 3261 cm −1 ) and ester groups (1721 cm −1 ), as well as aliphatic chains (779 cm −1 ). The compound 3 molecular ion peak was identified at m/z 497 [M+H]+, which, according to its mass and 13     tachment of the second sugar unit at C-2′ was suggested by the existence of the C-2′ carbon signal in the deshielded area at δ 83.39. No signal was seen after δ 4.04 in the 1 H NMR spectrum or from δ 171.52 to 103.27 in the 13 C NMR spectrum, indicating that the molecule was already saturated. Compound AB-04 acid hydrolysis generated palmitic acid and glucose, which were equivalent when analyzed using thin-layer chromatography (TLC). Based on this evidence, the chemical structure of compound 4 was determined as n-hexadecanoyl-(2′→1′′)-β-D-glucopyranosyl-β-D-glucopyranoside (Scheme 4).  The AB-05 compound capryloyl tetra glucoside was a light brown crystal mass pro duced from an eluant of chloroform-methanol (9:1). It showed positive glycoside tests The hydroxyl groups (3425, 3397, 3265 cm −1 ), IR absorption bands, as well as ester groups (1723 cm −1 ), and aliphatic chains were visible (779 cm −1 ). Based on the mass and 13 C NMR spectra, it was determined that the peak of the molecular ion for compound AB-05 was located at 792 [M]+. This result was in accordance with the chemical formula of the acy tetraglycoside, which is C32H56O2. It was determined that the capryl group was connected to a tetra glycoside unit based on the ion peaks that occurred at m/   Figure 1 displays the anti-inflammatory effects of isolated phytoconstituents AB-01 to AB-05 at 5 and 10 mg/kg and Indomethacin (10 mg/kg) on carrageenan-induced rat paw. When carrageenan-stimulated rats were given Indomethacin in combination with AB-01, AB-02, AB-03, AB-04, and AB-05 at a dosage of 10 mg/kg, there was a statistically significant reduction (p < 0.05) in the volume of their paws. Compared to a higher dose of Indomethacin (10 mg/kg), the effects of AB-01 and AB-05 at 10 mg/kg were shown to be  Figure 1 displays the anti-inflammatory effects of isolated phytoconstituents AB-01 to AB-05 at 5 and 10 mg/kg and Indomethacin (10 mg/kg) on carrageenan-induced rat paw. When carrageenan-stimulated rats were given Indomethacin in combination with AB-01, AB-02, AB-03, AB-04, and AB-05 at a dosage of 10 mg/kg, there was a statistically significant reduction (p < 0.05) in the volume of their paws. Compared to a higher dose of Indomethacin (10 mg/kg), the effects of AB-01 and AB-05 at 10 mg/kg were shown to be more effective in lowering the volume of the swollen paw. The aggravation is linked to the pathophysiology of different diseases, such as joint inflammation, cancerous growths, gout, and vascular diseases. In traditional clinical frameworks, various medicinal plants treat pain and inflammation signs. We found that at 5.0 and 10.0 mg/kg dosages, the AB-01, AB-02, AB-03, AB-04, and AB-05 extracted from A. indicum extract had a calming and analgesic effect, respectively. Carrageenan acted as a phlogistic factor, increasing the synthesis of prostaglandins and bradykinins at certain periods [40].
All the drugs that were examined demonstrated a reduction in paw edema volume from 1 h to 5 h, and their anti-inflammatory effect was continued beyond 3 h. This analysis unequivocally showed that all examined medications may have had an effect that cooperated with the prostaglandins surge. Curiously, the effects of AB-05 on carrageenaninduced paw edema were consistent throughout the 1 h and 5 h time points. Inflammatory mediators such as prostaglandins and platelet-activating factors (PAF) were triggered by carrageenan, leading to inflammation. Histamine, 5-hydroxytryptamine, and kinin all arrived during the first stage (0-1 h), whereas the second stage (3-5 h) was linked to the production of prostaglandin and bradykinin. Even though the 10 mg/kg dose had a greater calming effect than the 0.05 mg/kg dose, this finding was directly connected to the effects shown in carrageenan-induced paw edema, where both dosages of isolated compounds markedly changed the inflammatory response triggered by carrageenan [41]. The abdominal constrictions seen following the acetic acid treatment were related to the prostaglandin-induced sensitization of nociceptive receptors in assessing analgesic potential. Therefore, potential consequences of the concentrates may exert their painrelieving effects by impeding prostaglandins synthesis.
In the ADMET studies, QPlogHERG values for compounds AB 01 and AB 04 were less than the specified value, indicating they possess some cardiotoxic potential. Predictions of the Qikprop (version 5.1, Schrödinger, LLC, New York, NY, 2020) tool suggested that the non-active transport of these compounds across the gut-blood barrier was poor except for compound AB 01, with complete oral absorption. This was also the only compound capable of crossing the blood-brain barrier, as suggested by its QPlogBB and QPlogMDCK values. All compounds had some degree of skin permeability. As per Lipinski's rule of five, compound AB 01 exhibited only one violation; compounds AB 02 and AB 03 showed two violations, while compounds AB 04 and AB 05 displayed three out of five (Table 1). Hence, all data obtained from the predictions of the Qikprop tool gave an idea that compound AB 01 might serve the purpose of a potentially druggable compound. The isolated compounds AB-02, AB-03, AB-04, and AB-05 were docked with several receptors linked to inflammatory pathways. These receptors included cyclooxygenase-2 and tumor necrosis factor-alpha. Comparing the docked poses to the crystal conformation of each ligand revealed a rootmean-square deviation (RMSD) of 2.0 A • , further establishing the reliability of the selected docking method.

Conclusions
The phytochemical components of A. indicum, collected from the banks of the Koshi River in India, were depicted in this study. As a result, this study can significantly advance our understanding of the phytoconstituents in this herb and pave the way for the creation of novel medications with enormous promise for use in traditional Indian medicine. The current investigation suggests that AB-01, AB-03, and AB-05 show considerable analgesic and anti-inflammatory effects at 10 mg/kg. The main trigger for the release of platelet initiating factors (PAF), other inflammatory mediators and prostaglandins was carrageenan-induced edema. In conclusion, 5 -hydroxyhexyl n-hexadecanoate as AB-01 and n-octanoylβ-D-glucopyranosyl-(2 -1 )-β-D-glucopyranoside as AB-05 were isolated from A. indicum extract. These compounds showed anti-inflammatory and pain-relieving properties and may help to treat systemic inflammation caused by endotoxin. Since the precise purity of the extracted compounds has not yet been established, a potential contribution of minor compounds to the reported bioactivities cannot be completely ruled out. To the best of our knowledge, this work is the first to examine the presence of capryloyl tetra-glucoside and aliphatic ester in a methanolic extract of A. indicum. The study confirms that these strong isolated chemicals can be a useful diagnostic tool for the genus A. indicum. These results are thought to be crucial in nurturing the powerful compounds AB-01 and AB-05 for treating disorders caused by inflammation.

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.