The Agilent 1,290 Infinity UHPLC system, which was coupled to the Agilent 6,520 Accurate-Mass Q-TOF mass spectrometer, was used in conjunction with the Agilent Eclipse XDB-C18 column, which had dimensions of 2.1 × 150 mm and a particle size of 3.5 m. Throughout the analysis, the column temperature was held constant at 25 °C, while the auto-sampler temperature was set and held at 4 °C [18]. Mobile phase A, which flowed at a rate of 0.5 mL/min and contained a 0.1% formic acid solution in water, was chosen for the analysis. This was chosen in preference to another mobile phase (mobile phase B), which was a 0.1% solution of formic acid in acetonitrile. The analysis was performed for 25 min after infusing 1.0 µL of plant extracts that had been diluted in methanol of high-performance liquid chromatography (HPLC) grade. A post-run time of 5 minutes was noticed after the run. Nitrogen gas was used to create nebulization at a start-up flow rate of 25 L per hour. This was followed by increasing the flow rate to 600 L per hour to facilitate drying at 350 °C. The fragmentation voltage was kept at 125 V, while the capillary voltage was set at 3500 V (Saleem et al., 2019). Similar to previous findings, RP-UHPLC–MS analyses were carried out to evaluate the polyphenolic composition of TDME. Agilent MassHunter Qualitative Analysis B.05.00 software was used to evaluate the data using the Metabolomics-2017–00004. m method. Utilizing particular criteria, such as a match tolerance of 5 ppm, the compounds were found using the exploration database METLIN_AM_PCDL-Ne 170502 cdb. Both negative ion modes like H- and positive ion modes like H+, Na+, and NH4+ were employed (Locatelli et al., 2017).

A Shimadzu HPLC system from Japan was used to quantify the sample extract (Al-Qahtani et al., 2023). To determine the phenolic profile, a dried sample weighing 0.1 g was combined with 1 mL of methanol. The sample was filtered through a 0.45 μm syringe filter and centrifuged at 6,000 g for 15 min. A 10 µL volume was then injected into the HPLC apparatus after that. The Shimadzu Shim-pack CLC-ODS C-18 column (with dimensions of 5 cm × 4.5 mm and a particle size of 5 m) was used to analyze the samples. The standards used in this analysis for the quantification of polyphenols are chlorogenic acid, p-coumaric acid, gallic acid, ƿ-hyydroxy benzoic acid, caffeic acid, vanillic acid, kaempferol, sinapic acid, ferulic acid, salicylic acid, coumarin, quercetin, benzoic acid, and rutin. The calculation of the k-factor was used. The limit of detection and limit of quantification of these compounds were in the range of 0.15–0.46 and 0.42–2.47 μg/mL, respectively. Glacial acetic acid (1% in water), which was solution A, and methanol B were combined in the mobile phase at the volumetric ratios of 90:10, 84:16, 72:28, 65:35, 50:50, and finally, to 90:10 (acetic acid:methanol) at time intervals of 0–5, 6–20, 21–35, 36–45, and 46–60 min, and the final concentration in the mobile phase was run for 61–75 min to restore the original conditions, before the new sample was injected. The flow rate was maintained at 0.8 mL/min, and the injection volume was 10 µL. An RS Diode UV–VIS matrix detector was used to detect the signals of chromatogram and operated in a wavelength range of 270–370 nm. The temperature of the column was maintained at 25 °C, and identification of peaks was performed by the comparison of peaks obtained by running the standard compounds under similar conditions. The similar procedure was also reported by Basit et al. (2023). The experiment was performed in triplicates, and the obtained chromatogram is shown in Figure 1. Concentration of compound  µ g / g = Area of the Peak  *   k ‐ factor of the compound .

The method outlined earlier was utilized to measure the hemolytic impact of the TDME. A total of 10 mL of human blood was obtained from volunteer participants and placed in a sterile EDTA tube with a screw top. The tube was then put in a centrifuge and spun for 5 min at an acceleration of 850×g. After removing the top layer, the erythrocytes were carefully washed with 10 mL of sterile, cooled, isotonic PBS (pH 7.4). The previously cleaned erythrocytes were then reconstituted in 20 mL of sterile, cold PBS. The extracts were then added to the erythrocyte solution at a concentration of 1,000 μg/mL, and the mixture was then incubated at 37 °C for 60 min. The absorbance of hemoglobin in the supernatant at 540 nm was measured to estimate the rate of hemolysis. The proportion of hemolysis was computed using the stated formula. Triton X-100 (0.1%) was used as the +ve control, and PBS was used as the -ve control (Tabassum et al., 2022). Hemolysis percentage  % = Absorb .   of   sample   ‐   Absorb .   of   negative   control Absorb .   of   positive   control × 100 .

The technique of stabilizing the membrane of the human red blood cell was exploited in this study. Healthy human volunteers’ blood samples were combined with an equal amount of Alsever’s solution (5 mL blood sample and 5 mL Alsever’s solution). Alsever’s solution consists of 2% dextrose, 0.8% sodium citrate, 0.05% citric acid, and 0.42% sodium chloride. The mixture of blood sample and Alsever’s solution was formerly centrifuged at room temperature for 15 min at a speed of 3,000 rpm. These RBCs were washed with saline solution to eliminate impurities and to prepare a 10% suspension of RBCs. At the time of the experiment, different preparations were made: a control using distilled water and a standard solution of diclofenac sodium (at a conc. of 500 μg/mL and various concentrations of TDME at 500, 1,000, and 3,000 μg/mL). Distilled water, 1 mL of phosphate buffer, 2 mL of hyposaline (0.36%), and last, 0.5 mL of the red blood cell (RBC) suspension were added to each of these preparations in that order. The study followed the guidelines set in the Helsinki Declaration and received approval from the institutional ethical committee known as PREC. The assay combination was centrifuged for 20 min at a speed of 3,000 rpm following a 30-min incubation at 37 °C. At 560 nm, the supernatant solution’s hemoglobin content was calculated by spectrophotometry (Chirumamilla et al., 2022). %  inhibition = 1   –   Absorbance of sample /  Absorbance of control *   100 .

All of the materials utilized in the study were highly pure and appropriate for scientific investigation. Sigma-Aldrich (United States) provided carrageenan, Novartis Pharma Ltd. provided diclofenac sodium, and Indus Pharma provided pentazocine (Javed et al., 2020).

TDME (at dosages of 30, 100, and 300 mg/kg), pentazocine (at a dose of 10 mg/kg), diclofenac sodium, and indomethacin (at a dose of 15 mg/kg) were produced and dissolved in distilled water. These solutions were given intraperitoneally at a dosage of 5 mL/kg, acting as reference substances for assessing their anti-inflammatory and analgesic effects (Javed et al., 2020).

The Wistar albino rats were housed and cared for in the animal facility of the Pharmacology & Physiology research laboratory in Pakistan (the Islamia University of Bahawalpur’s Faculty of Pharmacy and Alternative Medicine). The rats were both male and female, weighing between 140 and 225 g. In this investigation, polycarbonate cages with a maximum animal occupancy of six were used, measuring 47 × 34 × 18 cm³. Standard environmental parameters were upheld throughout the trial, including a temperature of 25°C ± 2 °C, humidity levels of 50%–55%, and a consistent 12-h cycle of light and darkness. Throughout the trial, the animals received standard animal feed and had full access to water. The experimental circumstances were kept up for a week before the trial began to reduce stress on the animals. Under the reference number PAEC22/74, the institution’s Pharmacy Research Ethics Committee (PREC) authorized the study’s protocols and procedures (Basit et al., 2022).

An acute toxicity experiment was used to evaluate the safety of the TDME, with a few minor adjustments made based on the previously published research (Lalitha et al., 2012). Five groups of Wistar albino rats, each with five rats, were constructed, having both males and females in each group. The rats were given plenty of food and drink before the trial began, and they also got used to the laboratory environment. The rats that had been fasting for 12 h were given TDME at increasing doses (0.3, 1, 3, and 10 g/kg). The intraperitoneal (i.p.) method was used to administer the extracts. The study also included a control group that received the normal saline solution (10 mL/kg) for comparison. For the first 12 h, the rat was monitored every 1 h and then daily for the next 14 days. The mortality rate was measured after 48 h, and various physical and behavioral signs were identified and closely tracked in the animals. Tremors, convulsions, salivation, perspiration, lacrimation (tear production), the writhing reflex, somatomotor activity, and any notable behavioral abnormalities were among the symptoms.

A modified version of a previously established approach was used to examine the anti-inflammatory properties of TDME. For this assessment, the carrageenan-induced paw edema model was used (Basit et al., 2022). Thirty rats were taken. Six rats were included in each of the five groups from which the animals were separated. As a baseline measurement, the right hind paw of each rat was estimated with a screw gauge. The test drug, TDME, was given to separate groups of rats intraperitoneally (i.p.) at dosages of 30, 100, and 300 mg/kg. Distilled water was used as the administration method, and a dose of 5 mL/kg intraperitoneally was applied. Another group received a normal medication, indomethacin, at a dose of 15 mg/kg i. p. After giving the rats these drugs, 0.1 mL of a 1% carrageenan solution was injected into the plantar tissue of their right hind feet to cause edema. Before 1, 2, 3, and 4 h after the injection of carrageenan, the width of the paws was measured. The difference in paw thickness was calculated by deducting the first measurement (“0 h”) from the measurements collected at the following hourly intervals to calculate the growth in paw thickness (Vázquez et al., 1996).

The study used six groups of Wistar albino rats weighing between 150 and 200g and consisted of both males and females. Each group had six animals. The control group of rats received 5 mL/kg of distilled water intraperitoneally (i.p.). The control group obtained intraperitoneal (i.p.) injections of 15 mg/kg diclofenac sodium and 10 mg/kg pentazocine. The other groups received TDME intraperitoneally (i.p.) at dosages of 30, 100, and 300 mg/kg (Javed et al., 2020).

Computer-aided drug design and the advancement in molecular biology both benefit from the use of molecular docking. For molecular recovery, a technique for producing compounds as PDB files and a targeted search database in a valid Protein Data Bank (PDB) format is required. For this reason, many technologies such as the AutoDock Vina program, MGL Tools, Discovery Studio, PyRx, and Babel can be used. The PDB was used to find the receptor molecule used for the investigation. The responsibility for additional receptor preparation fell to Discovery Studio (Discovery Studio 2021 client). All the compounds screened by LC–MS were downloaded from PubChem as SDF files (structured data format). To make ligand compounds, the Babel was employed. Then, Vina, which was integrated into PyRx, was given the receptors and ligands. Specific target proteins were selected for the in vitro or in vivo assays involving them. α-Glucosidase (1obb) and urease (3la4) were used to find the relationship of compounds with in vitro inhibition properties of TDME. Lipoxygenase (6ncf) and cyclooxygenase (6y3c.COX1 and 6bl4. COX2) proteins were selected to evaluate the effect of compounds on in vitro and in vivo anti-inflammatory, analgesic, and antipyretic activities of TDME. The incorporation of tiny protein molecules into a target requires a planned sample of the ligand’s ability to precisely conform to a certain target pattern to produce an ideal complex form. By using the program evaluation function, this may be done. Vina was applied for docking, and Discovery Studio was used to display the findings of interactions from the docking (Meng et al., 2011).

On 28 June 2023, the absorption, distribution, metabolism, and excretion (ADME) features of bioactive compounds derived from reverse phase ultra-high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (RP-UHPLC–QTOF-MS) were evaluated by using SwissADME (https://www.swissadme.ch/) online tools, and these substances also underwent docking testing against several enzymes (Khan et al., 2023). Protox-II (https://tox-new.charite.de/) was used to evaluate the toxicity by following the reported procedure (Abdullahi et al., 2021).

The results of the current study showed good results of enzyme inhibitions (Table 4). The lipoxygenase (44.75 ± 0.51 µgmL^-1), α-glucosidase (52.71 ± 0.01 µgmL^-1), and urease (67.19 ± 0.68 µgmL^-1) inhibition showed significant results of TDME, which were comparable to the standards used (indomethacin, 18.03 ± 0.12 µgmL^-1; quercetin, 4.11 ± 0.01 µgmL^-1; thiourea, 8.97 ± 0.11 µgmL^-1). Our results were also in agreement with evidence by Sen et al. on lipoxygenase inhibition by the methanolic extract of another Typha species (Typha elephantina). Lipoxygenases (LOX) are enzymes that play an important part in the manufacture of leukotrienes, which are bioactive lipids. Their strength resides in their involvement in several physiological processes such as inflammation (Abbas et al., 2022), immune response control, and cell signaling, making them promising therapeutic targets in disorders such as asthma and other inflammatory ailments. Furthermore, the ability of lipoxygenases to catalyze lipid oxidation events increases their importance in lipid metabolism and overall cellular homeostasis (Sajid-ur-Rehman et al., 2023).

According to the previous study, T. domingensis is an excellent source of glucosidase inhibitors, iron chelators, and natural antioxidants. Oxidative stress is also a central consideration during the management of diabetes, and many bioactive phytochemicals including polyphenols are acknowledged as potent antioxidants (Ahmed et al., 2022b). The plant’s fruit extract is more effective as an anti-glucosidase and antioxidant than the male and female flower extract. In contrast, the female floral extract has the greatest ability to chelate iron among the three extracts. The phenolic content of the extract can be credited with both antioxidant and anti-glucosidase properties. Our research offers a molecular explanation for TDME’s capacity to treat diabetes and promote wound healing (Chai et al., 2014). This antioxidant potential is primarily due to the inhibition of pro-inflammatory mediator release, free radical neutralization, ROS, and RNS, which reduces lipid peroxidation and activates the cyclooxygenase pathway (Khatri et al., 2019). Additionally, they influence blood glucose levels by several methods, including limiting intestinal glucose absorption, promoting cell insulin production, and improving glucose uptake in insulin-sensitive tissue (Vinayagam et al., 2016).

Anti-urease drugs are now focusing on their potent anti-ulcer properties. Urease enzyme activity has been identified as a key pathogenic determinant in the etiology of several serious disorders that are harmful to human health, animals, and agriculture [67]. Sesquiterpene hydrocarbons or alcohols, or their synergistic action, may have very strong antibacterial urease activity. The primary defense against Helicobacter pylori may be due to urease inhibition, which prevents the bacteria from adhering to the gastric mucosa. The process of urease inhibition was discovered to be non-competitive, with both the substrate and inhibitor being incompetently attached to the enzyme.

Data on the hemolytic activity of the TDME are shown in Table 5. TDME has a hemolytic percentage value of 4.25 0.16%, and the cytotoxic positive control showed significantly high hemolysis (93.51% ± 0.36%). Previously, many methanolic extracts from plants were tested by the hemolytic activity to establish their possible safety, and our results fall in a similar range (Tabassum et al., 2023). The ability of some chemicals, such as toxins or enzymes, to cause the lysis or destruction of RBCs is referred to as hemolytic activity. This characteristic is useful in a variety of research and diagnostic applications, such as determining pathogen pathogenicity, researching the impact of certain substances on blood cells, and identifying specific medical problems associated with aberrant hemolysis. It can, however, be a negative component, creating health problems when detected in certain pathogenic agents or poisons. According to the reported methods, the hemolysis activity of less than 30% is considered safe and non-toxic for humans (Tabassum et al., 2022).

Water-induced lysis of RBCs was significantly inhibited by TDME (Table 5). The results of percentage hemolysis inhibition showed a value of 39.16% ± 1.33%, compared to the standard inhibitor of inflammation (diclofenac sodium) in this study, which showed an inhibition value of 55.91% ± 1.78% at the dose of 1,000 μg/mL. It was reported that the denaturation of protein cells and tissue injury are reflected in good correlation with inflammatory diseases [28]. This finding demonstrates the anti-inflammatory action of TDME. Several compounds identified in the current study (Table 1; Table 2; Table 3) may be responsible for this activity.

The TDME was shown to be non-toxic up to a dosage of 10 g/kg in acute toxicity experiments on rates to assess the potential negative effects of increasing doses of TDME. No poisoning symptoms were noticed, and no deaths were reported over the 48-h observation period.

All dose levels of TDME showed anti-inflammatory effects (Figure 4 and Table 6). Notably, after carrageenan administration, significant anti-inflammatory effects from TDME were observed during the third and fourth hours. The 300 mg/kg dose had the strongest effect during the fourth hour of the doses examined. Carrageenan is a potent inflammatory agent used to induce edema. Various inflammatory mediators are involved in the development of carrageenan paw edema, and that has been extensively used to assess the anti-edema effects of natural products [69]. In the current study, various phytochemicals, especially polyphenols (Table 1; Table 2; Table 3), may be contributing to the reduction of inflammation. Moreover, several phytochemicals identified by HPLC–MS also have reported anti-inflammatory effects (Table 1; Table 2), which also shows a probability of their participation in declining inflammation.

The crystal structures of α-glucosidase (PDB: 3WY1), urease (PDB: 1EJX), 5-lipoxygenase LOX (PDB: 6NCF), and cyclooxygenases (COX1, PDB: 6y3c and COX2, PDB: 1CX2) were obtained from https://www.rcsb.org/. The ligands were chosen from RP-UHPLC–MS, and their structures were downloaded from PubChem.

α-Glucosidase inhibitors are used for the treatment of type 2 diabetes to slow down the digestion and absorption of carbohydrates, helping to regulate blood sugar levels (Kashtoh and Baek, 2022). The ligands from RP-UHPLC–MS showed very good binding affinity against α-glucosidase. 1,3,4,5-Tetracaffeoylquinic acid (−9.2), deoxymiroestrol (−8.1), formononetin 7-O-glucoside-6″-O-malonate (−8.0), stipitatonate (−6.9), N-cyclohexanecarbonylpentadecylamine (−6.9), 4-methylumbelliferone (−6.8), nigerose (sakebiose) (−6.7), vanillic acid 4-sulfate (−6.5), emmotin A (−6.6), vanillic acid 4-sulfate (−6.5), nonic acid (−5.6), annuionone B (−5.4), L-galactose (−5.9), purine (−5.4), curcumenol (−6.4), theobromine (−5.7), ethyl 4-methylphenoxyacetate (−6.3), 3-eydroxy-3-methyl-glutaric acid (−5.7), and curcumenol (−6.4) are presented in Table 9. The higher negative values of the binding energy score represent the higher binding affinity of the ligand with the receptor molecule, which may be due to the chemical structures and interactions of compounds with amino acid residues (Ahmed et al., 2022a). The 2D interactions of α-glucosidase with ligands having the highest docking score are shown in Figure 7. The standard used for α-glucosidase was quercetin. The previous study also showed that the fruit and female flower extracts of T. domingensis are promising sources of glucosidase inhibitors (Chai et al., 2014).

Urease is used to determine the H. pylori infection, a common cause of gastritis and gastrointestinal ulcers. In-depth research on diverse classes of urease inhibitors has been provoked by the need to treat such illnesses (Kosikowska and Berlicki, 2011). A lot of ligands showed the strongest binding affinity like formononetin 7-O-glucoside-6″-O-malonate (8.7), deoxymiroestrol (−8.1), 1.3,4,5-tetracaffeoylquinic acid (−7.7), stipitatonate (−6.7), 6-(alpha-D-glucosaminyl)-1D-myo-inositol (−6.7), vanillic acid 4-sulfate (−6.6), codonopsine (6.5), l5-acetylamino-6-formylamino-3-methylurssacil (−6.4), emmotin A (−6.4), nonic acid (−4.9), annuionone B (−6.0), L-galactose (−5.5), purine (−5.1), gurcumenol (−6.7), theobromine (−5.4), ethyl 4-methylphenoxyacetate (−5.8), 3-eydroxy-3-methyl-glutaric acid (−5.0), and nigerose (6.3), as presented in Table 9. The 2D interactions of ligands with the highest docking score against urease are presented in Figure 8.

Lipoxygenase (LOX) is an oxidoreductase enzyme that is found in both plants and animals. Its enzymatic action results in the formation of aromatic molecules, making it useful in the production of pleasant fragrances. Furthermore, LOX regulates the synthesis of volatile compounds, making it a great natural taste enhancer in the food preparation process (Steele et al., 2000). The binding affinity of deoxymiroestrol (−8.6), N-cyclohexanecarbonylpentadecylamine (−7.3), emmotin A (−7.0), and vanillic acid 4-sulfate (−7.4) showed good results. Indomethacin standard was used for LOX. Formononetin 7-O-glucoside-6″-O-malonate and 1,3,4,5-tetracaffeoylquinic acid showed better binding affinity as compared to the standard compound. The 2D interactions of ligands with the highest docking score against lipoxygenase are presented in Figure 9.

Cyclooxygenase COX-1 inhibitors are used for the reduction of inflammation and also anti-platelet properties. Cyclooxygenase COX-2 inhibitors are used to alleviate pain and inflammation while minimizing gastrointestinal side effects compared to traditional nonsteroidal anti-inflammatory drugs (NSAIDs) (Ahmad et al., 2018). The binding affinity of deoxymiroestrol (COX-1, -8.0 and COX-2, -8.7), N-cyclohexanecarbonylpentadecylamine (COX-1, -7.0 and COX-2, -8.3), emmotin A (COX-1, -7.4 and COX-2, -7.3), and vanillic acid 4-sulfate (COX-1, -6.5 and COX-2, -6.8) is shown. The standard used for cyclooxygenase was indomethacin. Many compounds showed results higher than standard. The 2D interactions of ligands with the highest docking score against COX-1 and COX-2 are presented in Figure 10 and Figure 11, respectively.

All dots showed within pink color which is near to target. So, this is called an optimized drug. Smaller TPSA values (usually below 140–160 Å²) are frequently chosen for oral bioavailability in drug development, for instance, as bigger TPSA values may result in decreased permeability through cell membranes. On the other hand, compounds designed to engage with particular targets or receptors may benefit from having higher TPSA values (over 80–100 Å²). Typically, the molar refractivity range is 0–100 cm³/mol. The range of consensus Log P o/w is between −3 and +6. The solubility range is < −10 < poorly < −6 < moderately < −4 < soluble < -2 very <0 < highly. Skin permeation range is −1.2 to +1.32. If any compound follows at least three rules of drug-likeness, it can act as an oral drug (Shahid et al., 2023). To determine pharmacokinetic drug features, such as absorption, distribution, metabolism, and excretion, the Lipinski rule makes use of certain physicochemical qualities. The product’s molecular weight should not exceed 500 Da. The number of atoms in its molecules should range between 20 and 70, with an average of 50. The polar surface area must be less than 140 Å². Furthermore, the product should not contain more than five hydrogen bond donor sites and not surpass 10 hydrogen bond acceptor sites (Lipinski et al., 2012). The results of ADME and toxicological studies are presented in Table 10 and 11, respectively.