Evaluation of Senna tora (L.) Roxb. leaves as source of bioactive molecules with antioxidant, anti-inflammatory and antibacterial potential

Senna tora (L.) Roxb. is an ethno-medicinal herb used by rural and tribal people of the Satpura region of Madhya Pradesh in India and the Phatthalung Province of Thailand for treating rheumatism, bronchitis, ringworm, itches, leprosy, dyspepsia, liver disorders and heart disorders. It is also used in Chinese and Ayurvedic medicine. This study was conducted to investigate the potential of Senna tora (L.) Roxb. as a source of drug candidates against oxidants, inflammation, and bacterial infection. Preliminary phytochemical screening (PPS) and GC-MS were performed to identify the phytochemicals in the ethyl acetate extract of Senna tora (L.) Roxb. leaves (EAESTL). The in vitro antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH)- and H2O2-scavenging tests; the in vitro anti-inflammatory activity was determined by bovine serum albumin (BSA) denaturation and red blood cell (RBC) hemolysis inhibition; and the antibacterial activity was evaluated by agar-well diffusion methods. Cytotoxicity was estimated by Artemia salina larvae lethality, while acute toxicity was evaluated by oral delivery of the extract to mice. In silico antioxidant, anti-inflammatory, and antibacterial activities were predicted by the Prediction of Activity Spectra for Substances (PASS) program. The pharmacokinetics related to ADME and toxicity tests were determined by the admetSAR2 and ADMETlab2 web servers, and drug-able properties were assessed by the SwissADME server. GC-MS detected fifty-nine phytochemicals that support the types of compounds (phenols, flavonoids, tannins, terpenoids, saponins, steroids, alkaloids, glycosides and reducing sugar) identified by phytochemical screening. EAESTL exhibited dose-dependent antioxidant, anti-inflammatory, and antibacterial activities without any adverse effects or fluctuations in body weight. The PASS program predicted that the identified phytochemicals have antioxidant, anti-inflammatory and antibacterial activities. Among 51 phytochemicals, 16 showed good ADME, and 8 fulfilled drug-able properties without toxicity. Altogether, four phytochemicals, viz., benzyl alcohol, 3-(hydroxy-phenyl-methyl)-2,3-dimethyl-octan-4-one, phenylethyl alcohol and 2,6,6-trimethylbicyclo [3.1.1] heptane-3-ol, showed good pharmacokinetics and drug-able properties without toxicity, along with antioxidant, anti-inflammatory, and antibacterial activities. The obtained results suggest that Senna tora (L.) Roxb. leaves contain bioactive phytochemicals that have the potential to fight against oxidants, inflammation, and bacterial infection as potential drug candidates.

chloroform, filtered to collect the clear part, and transferred to 2 test tubes evenly. Afterwards, H 2 SO 4 (conc.) was added at the tube edge without shaking, and a bluish-brown layer at the interface suggested the presence of terpenoids. Concentrated H 2 SO 4 was added to another tube of EAESTL and agitated; after settling, a black color appeared on the bottom face, indicating that steroids were present in the sample. In Mayer's test, 25 mg of EAESTL was dissolved in 10 mL of aqueous HCl (1%) with constant stirring in a water bath and then filtered off to collect the clear part. 1 mL of Mayer's reagent was mixed with 1.5 mL of this clear acid-treated EAESTL solution. A creamy-colored precipitate appeared at the edge, indicating the presence of alkaloids in EAESTL [25]. To detect cardiac glycosides, legal tests were utilized. 5 mg of EAESTL was treated with 3 mL of pyridine, and then 2 drops of nitroprusside and a drop of 20% sodium hydroxide solution were added to this solution. The appearance of a blood-red color demonstrated the existence of cardiac glycosides in EAESTL [24. Frothing and foaming tests were used to identify the presence of saponins in EAESTL [24]. Here, distilled water (3 mL) was added to 0.5 mg/mL of EAESTL in methanol and shaken for a few min before being allowed to stand. Even after 10 min of standing, the foam did not disappear, demonstrating the presence of saponins. In Fehling's test, 5 mg of EAESTL was added to an equal volume of Fehling's solutions A and B and boiled for a few min. Reducing sugar was identified by the sudden appearance of a yellowish color, which later turned into a brick-red precipitate [24].

Gas chromatography-mass spectrometry (GC-MS) analysis
GC-MS was conducted in accordance with a previously described method [27] with minor modifications. A gas chromatography-mass spectrometer (Shimadzu triple-quad GCMS-TQ8040) was used to identify the phytochemicals in EAESTL. Helium gas and an Rtx-5MS capillary column (30 m × 0.25 mm id, film thickness of 0.25 m) were utilized as the mobile and stationary phases, respectively. The column oven temperature was controlled by dedicated software, and graduated heating was set at 50, 200, and 300 • C at 1, 2, and 7 min, respectively. The temperature of the sample injector was 250 • C, with a 40-min experimental run time.
This instrument was operated in splitless mode, where a 1 μL sample was injected. The constant flow rate was set at 1 mL/min. The detector parameters for GC-MS analyses were set as follows: 250 • C interface temperature, 230 • C ion source temperature, 50-600 (m/z) scanning range, and an ionization energy of 70 eV with a scan time of 0.3s. The phytochemical ingredients were identified by comparing the retention time and obtained pattern of the spectrum, which was then matched with the database of the National Institute of Standards and Technology (NIST). The individual peak area of an ingredient with respect to the total area of all other peaks was used to calculate the individual content (%) of an ingredient in EAESTL.

Antioxidant activity assay 2.8.1. In vitro antioxidant activity assay by scavenging DPPH free radicals
DPPH was used to determine the free radical-scavenging activity of EAESTL by following a previously described method [24]. Individual stock solutions of DPPH (1.5 mg/mL), EAESTL (10 mg/mL), and ascorbic acid (10 mg/mL) were prepared in methanol. A DPPH solution (60 μL at 1.5 mg/mL) was added to different concentrations (200,180,160,80,40,20,10,5, and 2.5 μg/mL) of EAESTL and ascorbic acid separately to a final concentration of DPPH 30 μg/mL and a final volume of 3.0 mL with methanol. The control was a 3.0 mL DPPH solution of 30 μg/mL concentration in methanol. The reaction mixtures were properly mixed and shaken, followed by 30 min of preservation in the dark. The absorbance was measured at 517 nm using a UV-vis spectrophotometer. For each analysis, three independent samples were prepared, and the average absorbance was used for the calculation. The free radical-scavenging activity was estimated utilizing the following formula (Eq. (1)): % free radical-scavenging activity = ( Abs control -Abs sample or Abs ascorbic acid ) where Abs control and Abs sample are the absorbance of the control and EAESTL-added samples, respectively. Abs ascorbic acid represents the absorbance of the standard sample, where ascorbic acid was added instead of EAESTL. Microsoft (MS) Excel was used to determine the IC 50 of EAESTL and ascorbic acid, where the percent inhibition was plotted with the respective concentration of the sample. The formula y = ax + b was used in the final calculation, where x is the IC 50 and was calculated at the y = 50 position of the curve.

Scavenging of hydrogen peroxide (H 2 O 2 )
The antioxidant activity of EAESTL was measured by H 2 O 2 -scavenging assay by following the method described earlier [28]. 1.0 M of H 2 O 2 solution was prepared using phosphate-buffered saline (PBS, pH 7.4) and 10 mg/mL of EAESTL solution and 10 mg/mL of ascorbic acid solution were prepared in methanol as stock solutions. A total of 60 μL of a 1 M H 2 O 2 solution was added to varying concentrations (200,180,160,80,40,20,10,5, and 2.5 μg/mL) of EAESTL and ascorbic acid separately to obtain a final concentration of H 2 O 2 of 20 mM and a final volume of 3.0 mL with 2880 μL of PBS and the rest of methanol. The control was 60 μL of 1 M H 2 O 2 solution plus 2880 μL of PBS and the rest of the methanol for a final volume of 3.0 mL. Before measuring the absorbance data, the samples were kept for 10 min for settling purposes, and then the absorbance was recorded at a wavelength of 230 nm by utilizing a UV-vis spectrophotometer. The percent (%) H 2 O 2 -scavenging activity was estimated by the following equation (Eq. (2)): % H 2 O 2 -scavenging activity = ( Abs control -Abs sample or Abs ascorbic acid ) where Abs control is the absorbance of the negative control sample, and Abs sample is the absorbance of the sample, where EAESTL was added. Abs ascorbic acid is the absorbance of the ascorbic acid-added sample. The IC 50 values for EAESTL and ascorbic acid were calculated by following a method similar to that described above in Section 2.5, where the H 2 O 2 -scavenging activity was plotted against the concentration of the solution.

Heat-induced hemolysis of RBC assay
The RBC suspension solution (sparingly soluble) was prepared by following the previously explained method, with minor modifications [29]. The whole blood of a healthy human individual was collected in a heparinized centrifuge tube. After collection, the blood was centrifuged for 5 min at 3000 rpm and washed three times with an equal volume of normal saline (0.9% NaCl). The upper clear part was discarded from the top of the tube. The residue part was reconstituted as a 10% (v/v) RBC suspension with an isotonic sodium phosphate buffer solution (10 mM, pH 7.4). To prepare 1 L of a 10 mM sodium phosphate buffer solution, 1.379 g NaH 2 PO 4 , 1.419 g Na 2 HPO 4 and 9.0 g NaCl were used. The stock solutions of EAESTL and ibuprofen were prepared by dissolving them separately into DMSO to a concentration of 10 mg/mL. The RBC suspension (0.05 mL) was added to the varying concentrations (200,180,160,80,40,20,10,5, and 2.5 μg/mL) of EAESTL and ibuprofen separately to a final volume of 3.0 mL with an isotonic sodium phosphate buffer solution. The control samples contained 0.05 mL of RBC suspension plus the same amount of DMSO instead of the tested sample and an isotonic sodium phosphate buffer solution with a final volume of 3.0 mL. In a shaking water bath, these samples were incubated at 54 • C for 20 min with constant shaking. After centrifugation at 2500 rpm, the clear part was collected from the top of the centrifuge tube. The absorbance of these sample solutions was measured at a 540 nm wavelength by using a UV-vis spectrophotometer. The following equation (Eq. (3)) was used to estimate the percent inhibition of hemolysis: Here, Abs sample and Abs control are the absorbances in the presence and absence of the EAESTL sample in PBS, respectively. Similarly, the ibuprofen-added sample was also compared with the control sample. The IC 50 value was calculated by following a similar method described in Section 2.5, where the percent inhibition of erythrocyte hemolysis was plotted against the concentration of the respective sample.

BSA protein denaturation assay
This experiment was performed by following a previously reported method with a slight modification [29]. 0.12 mL of 1% BSA was added to the varying concentrations (200, 180, 160, 80, 40, 20, 10, 5, and 2.5 g/mL) of EAESTL and ibuprofen separately to obtain a final concentration of BSA 0.04% and a final volume of 3.0 mL with phosphate buffered saline (PBS 1×, pH 7.4). The control samples were prepared by mixing 0.12 mL of 1% BSA with the same amount of DMSO instead of the tested sample and 1× phosphate buffered saline to a final volume of 3.0 mL and a final concentration of 0.04% BSA. All the samples were incubated in a water bath for 15 min at 37 • C, followed by an additional 5 min at 70 • C. The samples were allowed to cool, and the absorbance was recorded at a wavelength of 660 nm utilizing a UV-vis spectrophotometer. The following equation (Eq. (4)) was used to estimate the percent inhibition of protein denaturation: Here, Abs control and Abs Test sample are the absorbance of the negative control and EAESTL or ibuprofen added sample at a wavelength of 660 nm, respectively. The IC 50 of EAESTL and standard (ibuprofen) sample was calculated by following the same method described above in Section 2.5, where the percent inhibition of BSA denaturation was plotted against the concentration of the test sample.

Agar well diffusion assay
The agar well diffusion technique [24] was used to evaluate the antibacterial activity of EAESTL. All the tested bacterial strains were sub-cultured first by the streak plate technique on nutrient agar petri-plates. A single colony was taken from each bacterial culture plate of an individual strain and incubated in 25 mL of nutrient broth medium at 37 • C in a shaking incubator at 250 rpm and cultured to the mid-log phase of absorbance at 0.4 at 600 nm wavelength. 100 μL of fresh mid-log phase bacterial culture of each strain was spread on an individual nutrient agar petri-plate by a glass spreader and dried for 20 min. The four wells were produced by utilizing a 6 mm sterile cork borer on each nutrient agar petri-plate containing bacterial inoculums. A stock solution of EAESTL (500 mg/mL) was dissolved in DMSO and serially diluted to prepare 250, 125, 62.5 mg/mL concentrations of EAESTL. 100 μL of EAESTL solution of an individual concentration was applied sequentially in an individual well of the nutrient agar petri-plate. As a positive control, an antibiotic disc (erythromycin) was placed in the middle of the nutrient agar petri plate. As a negative control, 100 μL of DMSO was also applied into the well of nutrient agar petri-plate containing bacterial inoculums. Then, the nutrient agar petri-plates were kept in a refrigerator at 4 • C for 30 min to allow the samples to diffuse properly in the nutrient agar medium, followed by incubation at 37 • C for 16 h. The antibacterial activity was estimated by the zone of inhibition (within a well diameter) on the agar plate. The antibacterial activity assay was performed in triplicate.

Minimum inhibitory concentration and minimum bactericidal concentration determination
The twofold serial dilution method was used to test the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EAESTL [30]. A 500 mg/mL stock solution was placed in an autoclavable 15-mL glass tube and serially diluted with LB broth to achieve 250, 125, 62.5, 31.25, 15.625, 7.812, 3.906, and 1.953 mg/mL. A 50 μL bacterial culture in mid-log phase (absorption at 600 nm reached 0.4) was transferred to each tube. The control tube contained only bacterial strains and not the plant extract. The culture tubes were incubated at 37 • C for 24 h, and the turbidity of the tubes was examined. The lowest concentration of EAESTL that inhibited visible bacterial growth was considered the MIC. To determine the minimum bactericidal concentration of EAESTL, 50 μL of each culture was subcultured on the surface of an LB agar plate at 37 • C for 16 h. MBC was recorded as the lowest concentration of EAESTL that did not permit any visible bacterial colony growth on the LB agar plate after the incubation period. The MIC and MBC assays were performed three times.

Test of cytotoxicity by Artemia salina larvae lethality bioassay
The cytotoxicity of EAESTL was assessed using Artemia salina larvae as a test organism [31]. Artemia salina eggs were purchased from an aquarium shop in Dhaka, Bangladesh, and artificial seawater was used for hatching purposes. To prepare the artificial seawater, 38 g NaCl salt was dissolved in 1 L water followed by 48 h of hatching at 25 • C with constant aeration and irradiation of light. Active Artemia salina larvae were taken from this hatching and shifted to another vessel that contained fresh artificial seawater. The EAESTL and vincristine sulfate were dissolved separately into DMSO to a final concentration of 10 mg/mL. Each 0.1 mL of these solutions was mixed separately with 9.9 mL of artificial seawater in a total volume of 10 mL of 100 μg/mL concentration, from which serially diluted solutions were made to obtain a series of concentrations of 50, 25, 12.5, 6.25, and 3.125 μg/mL. An identical control was prepared without adding test samples but by diluting 0.05 mL of DMSO in 4.95 mL of artificial seawater to a final concentration of 1% DMSO, which is the DMSO concentration used in test samples. Ten (10) Artemia salina larvae per vial that contained either test or negative control samples were accommodated and examined after 24 h using a magnifying glass. The data were recorded based on the number of surviving Artemia salina larvae per vial. The following equation (Eq. (5)) was utilized for the estimation of the mortality of Artemia salina larvae for each concentration: where N T and N L are the quantity of Artemia salina larvae taken and alive, respectively, after a 1-day incubation period. The LC 50 was quantified using a mortality-versus-concentration curve, where the LC 50 value was calculated at y = 50 by following a method similar to that described for IC 50 in Section 2.5.

Acute oral toxicity study
The Organization for Economic Co-operation and Development (OECD) (Test No. 425) was followed during the assessment of the acute toxicity of EAESTL on Swiss albino mice [32]. An acute oral toxicity study was performed according to the method described previously [24]. Forty-two (42) male mice were divided into seven groups (6 in each), namely, the g-250, g-500, g-1000, g-2000, g-4000, g-5000, and control groups. Before starting the experiment, the mice were starved for 3-4 h, and only water was allowed during this time. A stock solution of EAESTL (500 mg/mL) was dissolved in 2% DMSO in a 0.9% saline solution. Each mouse in the g-250, g-500, g-1000, g-2000, g-4000, and g-5000 groups received a single oral dosage that contained 250, 500, 1000, 2000, 4000, and 5000 mg/kg EAESTL, respectively. A blank dosage containing only 0.25 mL DMSO (2% in 0.9% saline) was given to each of the mice in the control group. Following treatment, mice were allowed to feed ad libitum and were monitored for the development of any symptoms of toxicity (vomiting, diarrhea, ataxia, and death) after dosage administration. These monitoring data were recorded after 3 h of dosage administration and then every 4 h for 1 day. For the remaining 13 days, data was recorded once a day. The mortality of mice was calculated after 48 h of oral dosage administration using the following equation (Eq. (6)): where NMT and NML are the number of mice taken at the beginning and alive 48 h after EAESTL administration, respectively. The LD 50 was calculated by following a similar method described above in Section 2.5. Here, the mortality was plotted with respect to the dose amount (concentration) taken in the relevant test sample. At the end of the experiment, mice were placed in a new cage with corn cob bedding and immediately euthanized by displacement of air with 100% carbon dioxide, followed by being burial in soil at an approved landfill at our university. This experiment was repeated three times.

Bodyweight measurement
The body weight of the selected mice (six mice in each group) groups (g-Control, g-250, g-500, g-1000, and g-2000) was measured by following a reported method [33]. In summary, the initial body weight was recorded just before the extract was administered. The body weight of the mice was monitored for 2 weeks and weighed after 3, 6, 9, 12, and 14 days of extract administration. The cumulative weight change was estimated by using the following equation (Eq. (7)): where CW INTV represents the cumulative weight for a particular group at a specific time and CW INT is the same parameter at the initial time of the experiment.

Computer-aided prediction of the biological activity of phytochemicals
The PASS online server assessed the possible biological functions of EAESTL phytochemicals in the context of biological activities [34]. Briefly, the name of each phytochemical was submitted to the PubChem server, and a canonical SMILES of this element was extracted into the PASS program (http://www.way2drug.com/PASSOnline/) to obtain the probable biological activity of a specific phytochemical. The outcome was obtained as a ratio of 'probability of being active (Pa)' and 'probability of being inactive (Pi)' in the context of Pa>0.3. A higher Pa for a phytochemical indicates a greater chance of having a particular pattern of biological activity.

Prediction of pharmacokinetics (ADME), toxicity and drug-able properties
Different properties of the identified phytochemicals were evaluated in terms of being ideal drug candidates. The pharmacokinetic properties were related to ADME (absorption, distribution, metabolism, and excretion), toxicity (hERG inhibitor, AMES toxicity, carcinogenicity, hepatotoxicity, and rat oral acute toxicity), and drug-able properties [physicochemical parameters (MW, HBAs, HBD, RTB, and TPSA), lipophilicity (cLogP), water solubility (LogS), drug-likeness (Lipinski's Rule of Five: RO5) and medicinal chemistry (synthetic accessibility)]. The AdmetSAR2 web server (http://lmmd.ecust.edu.cn/admetsar2/) [35] was utilized to anticipate absorption (human intestinal absorption = HIA, human oral bioavailability = HOB, p-glycoprotein inhibitors, p-glycoprotein substrates), distribution (BBB penetration), metabolism (CYP450 inhibitors and CYP450 substrates), and toxicity (hepatotoxicity). The canonical SMILES ID of each phytochemical was submitted into the admetSAR2 web server, and the result was recorded for a set of parameters. Plasma protein binding (PPB) (distribution), excretion (renal clearance), and toxicity were predicted by the ADMETlab2 server (https://admetmesh.scbdd.com/) [36]. To do this, the canonical SMILES ID of each phytochemical ingredient was submitted to the ADMETlab2 server, and the result was obtained for a selected set of parameters. However, the SMILES format data for identified phytochemicals were further uploaded to the SwissADME server (http://www.swissadme.ch/), and computational results for physicochemical properties, lipophilicity, water solubility, drug-likeness and a synthetic accessibility score were collected [37].

Statistical analysis
The experimental data are represented as the average ± STD of the replicates for all experiments except body weight measurements of mice after oral administration of different concentrations of extract. The statistical interpretation of tests was accomplished using one-way ANOVA by Origin Lab version 2018, and Bonferroni's and Tukey's post hoc tests were used for the tests where *p < 0.05 and **p < 0.01 are significant compared with control values.

Phytochemical analysis of ethyl acetate extract of Senna tora (L.) Roxb. leaves (EAESTL)
Phytochemicals are indispensable in plant defense and have been used as important raw materials for pharmaceutical manufacturing for centuries. Therefore, we tested the existence of phytochemicals in the ethyl acetate extract of Senna tora (L.) Roxb. leaves (EAESTL) (Supplementary Figure S1). EAESTL was found to contain phenols (flavonoids and tannins), terpenoids, saponins, steroids, cardiac glycosides and reducing sugars, as listed in Table 1 and Supplementary Figure S2.         Compound's probable biological activity (PBA) was determined in the aspect of only antioxidant, anti-inflammatory, and antibacterial activity and Pa>0.3. Here, AOx = Antioxidant, A = Anti-inflammatory, AI = Anti-inflammatory intestinal, AO = Anti-inflammatory ophthalmic, AB = Anti-bacterial, ABO = Antibacterial ophthalmic.

GC-MS analysis of phytochemicals and predicting their antioxidant, anti-inflammatory, and antibacterial properties using PASS program
EAESTL had 59 peaks in its GC-MS chromatogram (Supplementary Figure S3). Each peak represented the existence of individual phytochemicals based on retention time (RT), peak area (%), chemical formula, compound structure and in silico, in vitro and/or in vivo biological properties ( Table 2). The major phytochemicals identified in EAESTL were esters [methyl hexadecanoate (PN53 in Table 2

DPPH and hydrogen peroxide radical scavenging activity
DPPH is a nitrogen-extractable oxidant that is conventionally used to determine the free radical-scavenging activities of antioxidants present in plant extracts or synthetic compounds. EAESTL at concentrations of 2.5, 5, 10, 20, 40, 80, 160, and 200 μg/mL exhibited dose-dependent antioxidant activity (Fig. 1A), with a range from 33.041 ± 1.166% to 91.068 ± 1.950% DPPH inhibition and an IC 50 value of 24.425 μg/mL. Similar antioxidant activity was shown by ascorbic acid, ranging from 40.455 ± 2.019% to 98.190 ± 0.863% DPPH restraint with an IC 50 value of 2.585 μg/mL. Although H 2 O 2 is not a free radical, it plays a pivotal role in causing oxidative damage by transforming into a highly reactive hydroxyl radical (HO • ). Therefore, we also performed an H 2 O 2 -scavenging assay for EAESTL and ascorbic acid. This analysis found concentration-dependent antioxidant activity for EAESTL by scavenging H 2 O 2 with a range of 34.595 ± 1.104% to 93.734 ± 0.336% and an IC 50 value of 17.434 μg/mL (Fig. 1B). Moreover, ascorbic acid showed H 2 O 2 -scavenging activity comparable with that of EAESTL, and the range was 39.238 ± 2.040 to 99.154 ± 0.115% with an IC 50 value of 1.923 μg/mL. In summary, comparing the results obtained in different scavenging assays, EAESTL and a well-known antioxidant (ascorbic acid) showed similar antioxidant activity.

EAESTL exhibited potent anti-inflammatory activity
Inflammation is firmly related to pathophysiological processes. The quest for natural compounds and phytoconstituents that might prevent inflammation could be helpful for human health. In this study, EAESTL displayed dose-related anti-inflammatory activity by inhibiting RBC hemolysis and BSA denaturation. At different concentrations, EAESTL inhibited hemolysis of the RBC membrane within a range of 31.058 ± 3.145% to 89.029 ± 1.186%, where the IC 50 value was 28.309 μg/mL ( Fig. 2A). In addition, the BSA denaturation inhibition of EAESTL ranged from 32.617 ± 0.890% to 91.731 ± 0.949, and the IC 50 value was 22.980 μg/mL (Fig. 2B). The antiinflammatory activity of EAESTL was compared with ibuprofen, which commonly functions as an active ingredient in standard  50 3.216 μg/mL). Thus, similar efficiency for EAESTL as for a prominent anti-inflammatory drug, ibuprofen, can be noted.

EAESTL exhibited potential antibacterial efficacy against a number of microorganisms that cause disease in humans
Medicinal plants containing bioactive phytochemicals have been used as a starting point for antibiotic synthesis to treat infectious diseases. Therefore, we assessed the antibacterial potential of EAESTL. Our findings revealed that EAESTL, at rates ranging from 62.5 to 500 mg/mL, can prevent the proliferation of bacteria, with inhibition zones ranging from 14 ± 1 to 23 ± 1 mm for Gram-positive bacteria and 11.5 ± 0.5 to 22 ± 1 mm for Gram-negative bacteria (Supplementary Figure S4). This finding demonstrates EAESTL's antibacterial effectiveness across a broad range of microorganisms. As shown in Fig. 3, against all the bacteria tested, a greater inhibition zone was obtained at a concentration of 500 mg/mL. EAESTL exhibited the highest antibacterial activity with a 23 ± 1 mm inhibition zone against S. aureus at 500 mg/mL, and the lowest antibacterial efficacy was 11.5 ± 0.5 mm against P. aeruginosa at 62.5 mg/mL. P. aeruginosa was always at the lowest level in terms of the zone of inhibition, while the growth of S. aureus was inhibited more than that of any other bacterial type studied. Erythromycin (EM) was used as a standard during this study and showed antibacterial activity with inhibition zones extending from 24 ± 1 to 31.333 ± 0.577 mm, which is comparable with EAESTL at a concentration of 500 mg/mL. The MIC and MBC values for the EAESTL tested on the nine bacterial strains are shown in Supplementary Table S1. The values recorded for MBC were greater than those for MIC, ranging from 3.270 ± 1.133 to 6.541 ± 2.266 mg/mL.

EAESTL has been found to be safe in cytotoxicity and acute toxicity tests
The cytotoxicity of EAESTL was tested using an Artemia salina larvae lethality bioassay. In this study, all Artemia salina larvae survived under control conditions after 24 h of EAESTL treatment. The mortality of Artemia salina larvae was proportionally dependent   Fig. 4A, the cytotoxic effect (LC 50 ) of EAESTL was 35.246 μg/mL; however, under the same conditions for a standard anticancer drug (vincristine sulfate), it was 2.238 μg/mL, and up to 100 μg/mL is considered nontoxic. Therefore, the cytotoxic effect of EAESTL can be considered moderately cytotoxic. The general focus of the acute toxicity studies was on determining the safe acute doses. Acute toxicity is generally expressed by the LD 50 value, which denotes the amount of potential poisoning material given at once for a short-term (usually less than 24 h) interval that causes the death of 50% of a group of test animals. In this work, the LD 50 was determined for Swiss albino mice. Any behavioral changes were also monitored to identify additional symptoms. None of the mice died after 24 h (Fig. 4B); however, after 40-43 h of dose implementation, 11.11 ± 9.622%, 50 ± 16.666%, and 61.111 ± 9.622% of the mice died at doses of 2000, 4000, and 5000 mg/kg b. w., respectively. Mouse mortalities that appeared within 40-43 h and up to 14 days of EAESTL (>2000 mg/kg) administration were considered for computing the oral LD 50 . The EAESTL oral LD 50 value for the mice was 4263.906 mg/kg b. w. (Fig. 4C), which is more than two times  higher than the nontoxic limit (>2,000 mg/kg b. w.) [83] and can be employed for further evaluation as a therapeutic agent to examine the effect of EAESTL on life-related parameters. During a 14-day observation period, EAESTL at a dose of 2000 mg/kg b. w. caused no death, behavioral abnormalities, locomotor ataxia, or diarrhea. At a dose of 2000 mg/kg b. w. (Fig. 4D), no noteworthy effect on body weight was noticed, and weight loss did not appear with increasing doses. From the behavioral monitoring, the food intake or water consumption attitude was similar for both test and control mice. This result indicates that EAESTL does not change the expected behavior of participating animals.

Sixteen phytochemicals exhibited good ADME to be possible drug candidates
The ADME profiles of 51 phytochemicals that showed individual antioxidant, anti-inflammatory, and antibacterial potential in the PASS program were investigated ( Table 3). The bioavailability of a drug is greatly affected by the route of drug administration, such as human oral bioavailability (HOB) and human intestinal absorption (HIA). Therefore, HIA and HOB were calculated at different drug development stages. Among the 51 phytochemicals tested, 50 showed positive HIA, and 34 exhibited good HOB. In the gastrointestinal tract, P-glycoproteins (Pgp) affect drug absorption. Our findings demonstrated that 48 phytochemicals are noninhibitors and 51 phytochemicals are nonsubstrates of Pgp. Thus, the pharmacokinetic efficacy of phytochemicals is neither inhibited nor lowered by Pgp. Some medications pass through the BBB, while others do not. All of the tested phytochemicals (51) showed no BBB permeability, indicating non-CNS targets. The impact of plasma proteins on drug delivery is the largest. Only the free (unbound to plasma proteins) drug can reach the tissues and produce a therapeutic benefit. Drugs with limited PPB (<90%) are thought to be therapeutically effective. We determined that 23 out of 51 phytochemicals have optimal plasma protein-binding potential with less than 90% PPB. Drug metabolism reduces the bioavailability of a drug. The endoplasmic reticulum of the liver pertains to a group of enzymes called cytochrome P450 (CYP450) that are needed for xenobiotic detoxification. Most orally administered drugs were also metabolized by these enzymes. Drugs that repress CYPs can be hazardous because of the aggregation of coadministered medications or the actual medicine. We observed that 51 phytochemicals are noninhibitors of CYP4503A4, 45 are nonsubstrates of CYP4503A4, 51 are nonsubstrates of CYP4502D6, 48 are nonsubstrates of CYP4502D6, 51 are nonsubstrates of CYP4502C9 and 47 are nonsubstrates of CYP4502C9, indicating that these phytochemicals neither accumulate nor undergo metabolism in the liver and will be able to enter the systemic circulation for distribution throughout the body to exert their desired action. Pharmacokinetics also manifest how drugs reach their target sites of action and are excreted. One of the most important routes for eliminating a metabolized and unaltered chemical is through renal excretion. Substances that will become future drug-like compounds must have excretion qualities during the drug development process. We revealed that 30 phytochemicals have modest clearance potential and that 2 phytochemicals have a high clearance tendency. However, above all, among 51 phytochemicals, 16 phytochemicals (PN3, PN6, PN11, PN12, PN13, PN14, PN17, PN18, PN23, PN25, PN28, PN32, PN35, PN37, PN42 and PN43) showed good ADME.

The drug-able properties of phytochemicals
A comprehensive evaluation of drug-able properties and toxicity is required for effective drug development. These properties greatly influence the ADME of the drug candidates. In this study, the SwissADME server was used to quantify these properties (Table 4) for the identified phytochemicals of EAESTL. Lipinski suggested RO5, which states that the four basic physicochemical properties are MW (<500 g/mol), HBA ≤ 10, HBD ≤ 5, cLogP 1> and ≤5. These are all correlated with higher HOB. Our computational study for EAESTL found that 49 phytochemicals have an optimal MW (<500 g/mol), and all 51 phytochemicals have an appropriate number of HBA (≤10) and HBD (≤5). In addition, 27 phytochemicals have a standard assortment of cLogP (1> and <5). The ideal RB range is 0-11, while the highest border for effective absorption is 13. Forty-four phytochemicals contain RB attributed to the standard range. Compound absorption is also connected to TPSA, and a TPSA of ≤140 is associated with high compound absorption. All identified phytochemicals have a TPSA value of <106.97, indicating that they have high permeability. The LogS value, which is always preferably low, impacts drug solubility. Twenty-eight phytochemicals have a suitable LogS value range of − 4.0 to 0.5, indicating that they can cross the membrane bilayer. Toxicity prediction is essential in the current drug discovery process for assessing a compound's adverse effects on an animal and the environment. The hERG gene (human ether-a-go-go-related gene) generally codes for a potentialdependent potassium ion channel that controls heartbeat. Many drugs have been abandoned due to cardiotoxicity induced by hERG inhibition. Among the 51 phytochemicals, 49 are noninhibitors of hERG. An obstacle to medicinal agents is carcinogenicity, which becomes severe when linked with mutagenicity. The Ames test is the most popular way of determining mutagenicity. Forty-five phytochemicals of EAESTL were found to be nonmutagenic in an Ames test (Table 4). Liver toxicity is one of the most critical aspects of the medication development process. The hepatotoxicity test revealed that 50 phytochemicals are not hepatotoxic. Acute oral toxicity is an essential endpoint in medicinal research and environmental risk management. However, the experiments are tedious to conduct; therefore, in silico approaches have been developed as a replacement. The United States Environmental Protection Agency (EPA) has classified toxicity based on LD 50 , and we found that among 51 phytochemicals, 42 are not acutely toxic. One of the wellknown methods of evaluating a drug's drug-likeness is RO5. In this study, the SwissADME server was used to assess the compounds' RO5, and 36 phytochemicals did not violate this set of rules, indicating that they are drug-like. The synthetic accessibility of a drug candidate is an important factor; for this reason, computational and medicinal chemists consider this to be a basic objective in the early drug development stage. Compounds with a synthetic accessibility score (SAscore) ≥6 are challenging to synthesize, and those with an SAscore ≤6 are easy to synthesize. Forty-eight phytochemicals showed a favorable synthetic accessibility score of ≤6 and the desired properties. However, only eight phytochemicals (PN7, PN14, PN24, PN28, PN34, PN35, PN37 and PN46) exhibited optimal druggable properties without toxicity. Taken together, four phytochemicals (PN14, PN28, PN35, and PN37) passed ADME and Table 3 The ADME profiles of the 51 phytochemicals that showed antioxidant, anti-inflammatory, and antibacterial activities in the PASS program. Here, "+" and "− " means present and absent, respectively; I = Inhibitor; NI = Noninhibitor; NS = Non-substrate; S = Substrate.

Discussion
Plants generate secondary metabolites and utilize them to defend themselves against possible adversaries and environmental risks. These metabolites and their derivatives have been effective therapeutic agents from the beginning of human evolution and play a significant role in treating various illnesses [84]. Therefore, this study explored the in vitro antioxidant, anti-inflammatory, and antibacterial activities of EAESTL and identified the phytochemicals in the extract to discover phytochemical compounds by means of a computational study of pharmacokinetics (ADME), toxicity, drug-able properties, and the probability of biological activities and in vivo cytotoxicity and acute toxicity to aid in developing potential therapeutic agents against oxidants, inflammation, and bacterial infection.
In living organisms, ROS are created endogenously or exogenously. The external supply of antioxidants acts as either ROS scavengers or inducers of endogenous (enzymatic and nonenzymatic) antioxidative systems to fight against ROS. Natural antioxidants produced from medicinal and dietary plants are commonly used as therapeutic agents. Therefore, we determined the antioxidant activity of EAESTL by using DPPH free radical-and H 2 O 2 -scavenging activity methods. EAESTL solution converted purple-colored DPPH to a yellow-colored DPPH-H protonated substance. Thus, the absorbance of DPPH diminished with increasing EAESTL condensation, indicating the free radical-scavenging potency of EAESTL. Similar free radical-scavenging activity was reported for the ethyl acetate extract of Xylocarpus granatum leaves by Darmadi et al. [85]. H 2 O 2 is not a free radical but it is an electrically neutral ROS. It can generate a highly reactive hydroxyl radical, which severely damages adjacent biomolecules [86]. In addition, it is hazardous due to its mobility, typically passing through cellular membranes and arriving at the cell compartments far from the site of its origination [87]. Thus, the removal of H 2 O 2 is crucial to avoid oxidative damage. Our antioxidant activity results showed that EAESTL scavenges H 2 O 2 in a dose-related manner, which is consistent with the known antioxidant capacities of the ethyl acetate extract of Fructus ligustri lucidi [88] and the methanol extract of Glinus oppositifolius [89]. The DPPH and H 2 O 2 scavenging capacity of EAESTL may arise from the phenolics, terpenoids, vitamin C, vitamin E, and carotene in EAESTL.
During inflammation, leukocytes discharge lysosomal enzymes, and proteases are part of their protective roles, causing more damage to the tissue [90]. Preventing hemolysis of the RBC membrane can provide insight into anti-inflammation because the RBC membrane and the lysosome membrane are identical. The firmness of such a plasma membrane can prevent or postpone cytolysis, preventing the liberation of cytosolic elements and, consequently, preventing tissue injury and the inflammatory reaction. Protein denaturation is one additional reason for inflammation [91]. An aid that can inhibit protein damage could be helpful in the treatment of inflammatory disorders. The ability of EAESTL to prevent RBC lysis and BSA protein denaturation was assessed as part of the research into the process of treating symptoms of inflammation. EAESTL exhibited concentration-dependent anti-inflammatory activity by inhibiting RBC lysis and BSA protein denaturation. The results demonstrate that this anti-inflammatory effect is perhaps attributed to the presence of esters, phenolics, terpenes, hydrocarbons, ketones, and aldehydes in EAESTL. Similar anti-inflammatory activity has also been observed in the research conducted by Gunathilake et al. [29], which showed that extracts of Cassia auriculata, Passiflora edulis, Sesbania grandiflora, Olax zeylanica, Gymnema lactiferum, and Centella asiatica prevented RBC lysis and BSA protein denaturation.
The bacterial strains used in this study are infectious in nature and can induce diseases. For instance, B. infantis was found in a neonate with sepsis, and Exiguobacterium sp. promotes pneumonia (CAP) and bacteremia [92,93]. S. aureus is among the most prevalent human pathogens, exacerbating bacteremia, respiratory tract and soft tissue infections, and endocarditis [94]. Some Streptococcus sp. involved in the pathogenesis of upper respiratory infections [95], and virulent E. coli accelerate gastroenteritis and urinary tract infections [96]. V. cholerae is an enteric pathogen that releases cholera toxin and causes acute secretory diarrhea [97]. S. typhi is an MDR-resistant bacterial pathogen that usually causes typhoid and paratyphoid, and it has rapidly gained resistance to previously efficacious drugs, such as ciprofloxacin [98]. P. aeruginosa can induce significant life-threatening infections by preventing the immune systems of affected individuals [99]. H. influenzae is the main bacterial agent that spreads diseases in the respiratory and sensory systems of the body [100]. Therefore, the antibiotic activity of EAESTL was assessed against these bacteria. The presence of physiologically active chemicals such as phenolics, esters, terpenes, hydrocarbons, aldehydes, and ketones could account for this antibacterial action. Our findings are in accordance with the previous research data that plant extracts high in phenolic compounds [101], fatty acid methyl esters [102], terpenes, hydrocarbons [103], aldehydes, and ketones [104] have antibacterial activity. Our recent observations show that the ethyl acetate extract of Ruellia prostrata Poir. has significant antibacterial activity against B. infantis, Exiguobacterium sp., E. coli, and P. aeruginosa [24], suggesting a similar potential for inhibiting these pathogenic bacteria that share a common carnage mechanism. The study conducted by Saranraj et al. [105], in accordance with our results, found that an ethyl acetate extract of Acalypha indica plant leaves had antibacterial activity against bacterial strains of S. aureus, S. typhi, V. cholerae, and E. coli.
The unexpected side effects of a particular drug compound can be avoided by estimating the level of toxicity with respect to the dose. Therefore, the cytotoxicity and acute oral toxicity studies were performed in Artemia salina larvae and mice, and we found that the LC 50 and LD 50 of EAESTL were 35.246 μg/mL and 4263.906 mg/kg, respectively. The LC 50 value of EAESTL is lower than the LC 50 value of standard vincristine sulfate. The LD 50 value of EAESTL is beyond 2,000 mg/kg, which indicates that it is nontoxic. In the acute toxicity study, if the LD 50 value is above 2000 mg/kg, it is considered nontoxic [83]. Thereby, EAESTL can be a good source of potential therapeutic agents. Successful drug development entails an accurate evaluation and rationalization of pharmacokinetics (ADME) and drug-able properties. Therefore, we investigated these properties in silico and discovered that only four phytochemicals, namely, 2,6, 6-trimethylbicyclo [3.1.1] heptane-3-ol (PN14), benzyl alcohol (PN28), 3-(hydroxy-phenyl-methyl)-2,3-dimethyloctan-4-one (PN35), and phenylethyl alcohol (PN37), passed ADME and demonstrated good drug-able properties without toxicity. Among these four phytochemicals, 2,6,6-trimethylbicyclo [3.1.1] heptane-3-ol also known as isopinocampheol or 3-pinanol, a terpenoid in nature, was found to have anti-inflammatory and antibacterial activities. In accordance with our results, in vitro activity investigations of essential oils from Hyssopus officinalis [51], Cedrelopsis grevei leaves [106] and the hexane-ether extract of Tanacetum santolinoides [107] have been reported to contain 2,6,6-trimethylbicyclo [3.1.1] heptane-3-ol, which has anti-inflammatory and antimicrobial properties. Benzyl alcohol exhibited anti-inflammatory activity. Consistent with our findings, the chloroform fraction of aerial parts of Isodon rugosus Wall. ex Benth contains benzyl alcohol along with other bioactive phytochemicals that have been reported to have anti-inflammatory and analgesic activity when analyzed in vivo [58]. Both the ethanol extract of Albizia coriaria (Welw ex. Oliver) leaves and the ethyl acetate extract of Albizia coriaria (Welw ex. Oliver) stem bark contain benzyl alcohol as a significant component and are reported to have in vitro antioxidant and antibacterial properties [59]. 3-(hydroxy-phenyl-methyl)-2,3-dimethyl-octan-4-one was found to have anti-inflammatory activity. Consistently, an ethyl acetate extract of Ruellia prostrata Poir. aerial parts contains 3-(hydroxy-phenyl-methyl)-2,3-dimethyl-octan-4-one, a phenolic molecule with anti-inflammatory properties [24]. Phenylethyl alcohol displayed anti-inflammatory activity. Our findings are also in good agreement with the results reported by Dadkhah et al. [59] and Amor et al. [63] who observed that phenylethyl alcohol is one of the main phytoconstituents of Rosa damascena Mill. and Phlomis species essential oils and exhibited antioxidant and anti-inflammatory activities, respectively. In addition, phenylethyl alcohol is a driven compound of Trichoderma virens 7b hexane extract, which showed antifungal activity against Ganoderma boninense, the causal agent of a devastating disease affecting oil palm in Southeast Asian countries [60]. Collectively, our results may contribute to the development of antioxidant, anti-inflammatory, and antibacterial agents for humans. Moreover, this work also shows a correlation between the ethnomedicinal uses of Senna tora (L.) Roxb leaves and their pharmacological activities.

Conclusion
The findings of this study concluded that Senna tora (L.) Roxb. leaves contain antioxidant, anti-inflammatory, and antibacterial activities due to the presence of some biologically active phytochemicals, viz., phenolics, terpenoids, vitamin C, vitamin E, carotene, esters, hydrocarbons, ketones, and aldehydes. Based on pharmacokinetics, drug-able properties, and toxicity, this study also identified four phytochemicals including 2,6,6-trimethylbicyclo [3.1.1] heptane-3-ol, benzyl alcohol, 3-(hydroxy-phenyl-methyl)-2,3-dimethyloctan-4-one, and phenylethyl alcohol, as possible therapeutic candidates. Among the four phytochemical compounds, 2,6,6-trimethylbicyclo [3.1.1] heptane-3-ol is anti-inflammatory and antibacterial; benzyl alcohol is an antioxidant, anti-inflammatory, and antibacterial; 3-(hydroxy-phenyl-methyl)-2,3-dimethyl-octan-4-one is anti-inflammatory; and phenylethyl alcohol is both an antioxidant and an anti-inflammatory. Further research is needed for the isolation of these phytochemical compounds with fascinating in vitro and in vivo pharmacological properties and to assess their safety and bioavailability in in vivo animal models to aid in developing drugs against oxidants, inflammation, and bacterial infection.

Author contribution statement
Md. Mashiar Rahman and Shahina Akhter conceived and designed the experiments, contributed reagents, materials, analysis tools, or data, and wrote the paper. Md. Abdullah Al Noman, Shapla Khatun, Rahat Alam, Md. Mahade Hasan Shetu, Enamul Kabir Talukder, Raihan Rahman Imon, and Md. Yaman Biswas performed the experiments, analyzed and interpreted the data. K. M. Anis-Ul-Haque and Mohammad Jashim Uddin analyzed and interpreted the data and wrote the paper.

Funding
No external funding was received for this research work.

Declaration of competing interest
None.