Exploring the medicinal potential of Senna siamea roots: an integrated study of antibacterial and antioxidant activities, phytochemical analysis, ADMET profiling, and molecular docking insights

Nowadays, infectious diseases pose an alarming global threat to human health. The genus Senna is among the most well-known taxonomic categories commonly used in folk medicine to confront these challenges. Motivated by its traditional uses, a comprehensive study was conducted on the roots extract of Senna siamea , aiming to address the in vitro antibacterial and antioxidant efficacy of phytochemicals from the dichloromethane: methanol (1:1) roots extract of the plant, along with in silico computational studies. The separation of compounds was achieved using silica gel column chromatography. Whereas, the antibacterial and antioxidant activities were examined using paper disc diffusion and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays, respectively. Silica gel column chromatography of the dichloromethane: methanol (1:1) roots extract afforded lupeol ( 1 ), β -sitosterol ( 2a ) and stigmasterol ( 2b ), chrysophanol ( 3 ), betulinic acid ( 4 ), and glyceryl-1-hexacosanoate ( 5 ). Although these compounds have been previously reported from the plant, proof of their medicinal applications via in vitro and in silico studies is still lacking. Notably, our findings showed remarkable inhibition zones by the extract (18.00 ± 0.00 mm and 17.17 ± 0.24 mm) against E. coli and S. aureus , respectively, at 50 mg/mL compared to ciprofloxacin (23.33 ± 0.47 mm and 22.00 ± 0.00 mm, respectively), showcasing its potential antibacterial efficiency. Considerable inhibition zones were also recorded by chrysophanol ( 3 ) against E. coli (16.33 ± 0.24 mm) and S. pyogenes (16.00 ± 0.00 mm) at 2 mg/mL, compared to ciprofloxacin which showed 23.33 ± 0.47 mm and 21.67 ± 0.47 mm, respectively, signifying its potent antibacterial activities. In addition, the crude extract and chrysophanol ( 3 ) exhibited substantial IC 50 values (1.24 and 1.71 µg/mL, respectively), suggesting their significant antioxidant potential compared to that of ascorbic acid (IC 50 : 0.53 µg/mL). Chrysophanol ( 3 ) fulfilled Lipinski’s rule with no violation and lupeol ( 1 ), β -sitosterol ( 2a ), stigmasterol ( 2b ), betulinic acid ( 4 ), and glyceryl-1-hexacosanoate ( 5 ) displayed one violation each which were in favor of the drug-likeness predictions. All the compounds exhibited no cytotoxicity and except betulinic acid ( 4 ), all the compounds also showed no carcinogenicity properties which


Introduction
The genus Senna, also known as Cassia, belonging to the Fabaceae family, comprises more than 260 species found in Africa, Latin America, Southeast Asia, and Northern Australia.These species have attracted keen interest in phytochemical, biological, and pharmacological studies due to their vast therapeutic values [1].The genus includes a range of plants, from giant trees to small seasonal herbs, with the majority being herbaceous perennials [2,3].Species of the genus are well known in folk medicine to cure a variety of internal and external disorders, including cancer [4], malaria [2], diabetes [5], bacterial and fungal infections, and mutagens [6,7].In Brazilian traditional medicine, Senna species have been used as purgatives, laxatives, and treatment of flu and colds [8].In Ayurveda, these plants were employed against headache and fever and in Thai traditional medicine, the species have been used in the treatment of abdominal and skin infections [9].
Senna siamea Lam (synonym, Cassia siamea, commonly called the Kassod tree, Fig. 1) is widely grown in Africa, Oceania, and Latin America [10], with a wide spectrum of traditional uses.In Ethiopia, it is locally called Yefereng digita (Amharic name) and is used by traditional healers to treat dandruff, wounds, common warts, pyoderma, gonorrhea, anthrax, and snake bites [11].Infusion of the pods is suggested to heal fever and as a laxative for pregnant women.Decoction of the pods is recommended against breathing difficulties and to treat intestinal worms.In addition, an infusion of extracts from the leaves is drunk to cure nosebleeds, convulsions, and flatulence [12].More traditional claims have also been cited for Senna siamea to be used for the treatment of jaundice, abdominal pain, menstrual pain, typhoid fever, and ease of sugar levels in the blood [13].The ethno-medicinal applications of the plant also revealed that it is used to treat microbial infections and asthma [14], sleeplessness, hypertension, malaria, liver disorder, constipation, toothache, diabetes [15,16], digestive problems, herpes, genitourinary disorders, rhinitis, and as a blood cleaning agent [17].
Regardless of the subspecies, various parts of S. siamea have been evaluated for their antimalarial properties, a tropical endemic disease with high morbimortality and have displayed auspicious results [18,19].The compound chrobisiamone A isolated from the leaves extract displayed attractive in vitro antiplasmodial activity against Plasmodium falciparum 3D7, and lupeol and emodin identified from the stem bark of the plant exhibited the antimalarial properties [20].The stem bark extracts have been reported to possess anti-inflammatory and analgesic properties [21].The leaves show antilipemic and antidiabetic effects [22], antibacterial activities [23], and antiproliferative effects [24].Moreover, the roots and flowers exhibit antioxidant activities [25].Hu and co-workers reported antiviral chromones from the stem of S. siamea with anti-HIV-1 and antitobacco mosaic virus (anti-TMV) activities [26].Besides, cassiamin B reported from the plant exhibited chemo-preventing and antitumor-promoting effects [27].
Despite the abundance of S. siamea in Ethiopia, scientific reports on the ethno-medicinal benefits of compounds isolated from its roots are still lacking.Although traditional healers use the plant in folk and livestock remedies, the medications are outdated and delivered without scientific support.Most traditional practices of the were consistent with the prediction results of ciprofloxacin.The molecular docking computations revealed that all the compound isolates displayed strong and nearly strong binding affinities against all protein targets, ranging from − 6.6 kcal/mol to -9.2 kcal/mol (lupeol (1) against E. coli DNA gyrase B and topoisomerase II α, respectively).Thus, the present findings suggest the roots of Senna siamea for potential medicinal applications against multidrug resistant pathogens hence validating its ethno-medicinal uses.
plant in Ethiopia are related to wound healing and antibacterial, and antioxidant activities [11,15].In addition, in silico investigations such as pharmacokinetics, druglikeness properties, and molecular docking studies of the compound isolates should be addressed.Thus, considering its promising multiple biological activities, this study aimed to evaluate the antibacterial, antioxidant, in silico cytotoxicity, and pharmacokinetic properties of compounds isolated from the roots of S. siamea, along with their in silico molecular modeling studies.

Collection and identification of the plant material
Roots of S. siamea were harvested from Adama Science and Technology University campus, Adama, Ethiopia, and its surroundings in July 2022.Mr. Reta Regassa (chief botanist, Hawassa University) in collaboration with Mr. Melaku Wendafrash (chief botanist, Addis Ababa University) identified the plant.After identification, a voucher specimen (HSS006) was deposited at the National Herbarium, Addis Ababa University, Ethiopia.The fresh plant materials were freed from impurities with continuous washing using tap and distilled water consecutively, followed by air drying for three weeks at room temperature without direct exposure to sunlight.Thenceforward, the plant samples were crushed into a fine powder and kept in a polyethylene bag until extraction.

General experimental procedures
The plant materials were powdered using a grinder (Shanghai Jingke, JK-HSG-100 A, China).Solvents (analytical grade), reagents, spraying chemicals, and positive controls were purchased from local markets, in Addis Ababa, Ethiopia.The powdered samples were macerated and concentrated on an orbital shaker (Gemmy Industries, VRN-200, Taiwan) and rotary evaporator (DW-RE-3000, China), respectively.The concentrates were chromatographed on silica gel (200 g, 60-200 mesh) in a column (size; 725 mm) and eluted with an increasing gradient of solvents.The purity of fractions was analyzed using TLC (thickness; 0.25 mm, size; 20 × 20 cm coated with high-grade silica gel, 230-400 mesh, pore size 60 Å, Merck Grade 64271, Darmstadt, Germany) and UV lamp (UV4AC6/2, CBIO Bioscience and Technologies, China, at 254 and 365 nm).Conversely, UV inactive compounds were detected using a spray (1% vanillin in ethanol and 2 mL sulphuric acid reagent) followed by direct heating (110 °C) over a stove for 5 min.Melting points were measured using a Japson-type apparatus (JA90161, India) and antioxidant activities were determined using a UV-Vis spectrophotometer (CE4001, UK) equipped with tungsten and deuterium lamps.NMR spectra were generated with the help of 400 and 600 MHz Bruker AVANCE type NMR instruments with deuterated chloroform and TMS as a solvent and reference, respectively.For antibacterial activity, an autoclave, incubator, micropipettes (1000 µL), Petri dishes (90 mm), and a hood equipped with laminar airflow and UV radiation were utilized.

Extraction and isolation
The pulverized roots of S. siamea (700 g) were extracted with 3.5 L of CH 2 Cl 2 : CH 3 OH (1:1) over an orbital shaker for 72 h by maceration technique.Whatman No. 1 and a vacuum rotary evaporator were used for the filtration and concentration techniques, respectively.The crude extract (24.0 g) was adsorbed on silica gel (25 g, 60-200 mesh), subsequently subjected to silica gel column chromatography (200 g, 60-200 mesh), and eluted with an increasing gradient of EtOAc in n-hexane followed by methanol in CHCl 3 ratio.The fractionation process generated a total of 75 fractions (100 mL each) and their purity was monitored using TLC, a UV lamp, and a vanillin-sulfuric acid reagent.Similar fractions with identical R f values in the same solvent system were mixed and structural information of the purified compounds was established using 1D and 2D NMR spectroscopic techniques.Fractions 8-10 collected with 25% EtOAc in n-hexane were mixed and purified with a gradient elution of 100% n-hexane up to 50% EtOAc in n-hexane to afford lupeol (1, 12.6 mg) and a mixture of β-sitosterol (2a) and stigmasterol (2b) (23.8 mg).Fractions 15, 16, and 17 obtained with 40%, 45%, and 50% EtOAc in n-hexane, respectively, were combined and purified with a gradient elution of 20-70% EtOAc in n-hexane to yield chrysophanol (3, 10.3 mg) and betulinic acid (4, 13.8 mg).Furthermore, fraction 18, isolated with 50% EtOAc in n-hexane was purified with preparative TLC using 25% EtOAc in n-hexane as a mobile phase to afford glyceryl-1-hexacosanoate (5, 9.5 mg).

Antibacterial activity
The CH 2 Cl 2 : CH 3 OH (1:1) extract and the compound isolates were studied for in vitro antibacterial activities against four human bacterial pathogens (Escherichia coli, ATCC-25922; Staphylococcus aureus, ATCC-25923; Pseudomonas aeruginosa, ATCC-27853; and Streptococcus pyogenes, ATCC-19615) using paper disc diffusion assay.The activities were performed following the standard protocols adopted by Akhtar et [44][45][46] using a commercial antibiotic (ciprofloxacin) and DMSO as reference drug and solvent, respectively.Different concentrations of the crude extract (25, 12.5, and 6.25 mg/mL) and isolated compounds (1, 0.5, and 0.25 mg/mL) were prepared in 4% DMSO from the corresponding stock solutions (50 mg/mL for the extract and 2 mg/mL for the compounds).The sterilized paper discs (6 mm) transferred to the bacterial culture-inoculated MHA were impregnated with 100 µL of each solution using a micropipette.Afterwards, the Petri dishes were left for 30 min for complete diffusion and incubated at 37 °C for 18-24 h.The clear zones of the paper discs revealed inhibition zones (mm) which were measured by a caliper (mm).The experiment was performed in triplicate and the results are expressed as mean ± standard error of the mean (SEM) using a Microsoft Excel 2016 spreadsheet.

Radical scavenging activity
The antioxidant activities that are related to the ability of the extract and compound isolates to transfer electrons or hydrogen atoms by bleaching the purple color of the DPPH solution in methanol were screened via 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay method, following previous experimental protocol [47].Various concentrations of the extract, compound isolates, and ascorbic acid standard were prepared in methanol by serial dilution and fresh DPPH solution (2 mL, 0.04% w/v in CH 3 OH) was added to each solution.The sample solutions were left to stand in the dark at room temperature for half an hour and their absorbance was measured at λ = 517 nm using a UV-Vis spectrophotometer in triplicate.Finally, the scavenging activities were calculated as mean ± SEM using Eq. 1 and the IC 50 values were determined from the relationship curves.

Molecular docking studies
The modes of binding of the compound isolates to protein targets of the bacterial strains and human enzymes were studied using molecular docking experiments and compared to the reference drugs.Accordingly, DNA gyrase B of E. coli (PDB ID: 6F86), PK of S. aureus (PDB ID: 3T07), 10782 streptopain of S. pyogenes (PDB ID: 6UKD), human myeloperoxidase (PDB ID: 1DNU), and topoisomerase II α (PDB ID: 4FM9) were obtained from the Protein Data Bank (PDB) (http://www.rcsb.org)and saved in the PDB format.The appropriate 3D structures of the compounds were retrieved from PubChem and/or Zinc docking databases, downloaded in SDF format, converted to PDB format via the Open Babel GUI application, and saved in PDB format.Proteins and ligands were prepared as per previous protocols [51,52] and molecular docking was computed by studying the binding interaction of the ligand and the receptor on the active sites of the protein targets.The 3D sizes of the structure-based design (SBD) site sphere of each protein were retrieved as follows; PDB ID: 1DNU (36.727, 9.922, and 10.989), PDB ID: 6F86 (61.680, 28.330, and 64.290), PDB ID: 6UKD (-25.515,-13.743, and 13.171), PDB ID: 3T07 (0.013, 0.147, and − 0.003), and PDB ID: 4FM9 (17.245, 39.350, and 25.275) Å for x, y and z dimensions, respectively.The docking simulation was performed using the MGL AuthoDock Tools 1.5.6 program and all the files dropped to the appropriate working directory were computed via the Command Prompt application.Nine different conformations were generated and only a single conformation with the most stable binding affinity and root mean square deviation (RMSD) was chosen.Finally, the ligand interactions, hydrogen bonds, residual amino acid interactions, 2D and 3D structural orientations, and image preparations were run using the Biovia Discovery Studio Visualizer 2021 [53].
Compound 1 (12.6 mg, R f : 0.45 in 10% EtOAc in n-hexane) was obtained as white sparkles with a melting point of 212-214 °C which was in good agreement with the reported values for lupeol [37,54].S1; Fig. S1).The 1 H-1 H coupling of adjacent protons was also supported by the COSY correlations.Accordingly, important  S1; Fig. S2 and S3).Overall, the spectral data obtained were in close agreement with the reported values for lupeol (1) [37,55] (Fig. 2).The compound was previously reported from the stem barks of S. siamea [56].

Antioxidant activity
The antioxidant potentials of the CH 2 Cl 2 : CH 3 OH (1:1) roots extract and compounds were determined via DPPH radical scavenging assay.The extract showed a scavenging activity of 89.25 ± 0.00 and 73.50 ± 0.00% at 1000 and 62.5 µg/mL, respectively, with IC 50 value of 1.24 µg/mL compared to ascorbic acid (92.76 ± 0.28 and 77.38 ± 0.35% at the 1000 and 62.5 µg/mL, respectively, with IC 50 value of 0.53 µg/mL.Of the compounds, chrysophanol (3) exhibited the highest scavenging potential at 1000 µg/mL (85.59 ± 0.15%) and 62.5 µg/mL (70.07 ± 0.33%) with an IC 50 value of 1.71 µg/mL.Whereas, the smallest scavenging activity was displayed by lupeol (1) with an IC 50 value of 3.93 µg/mL (Table 2; Fig. 3).Antioxidants have been investigated to show cell protection from free radicals causing oxidative cleavage which avoids aging and cancer.They serve as oxygen scavengers by interacting with free radicals, thereby disrupting the oxidation process [69].Previous studies revealed that synthetic antioxidants loom to cause cancer diseases.Consequently, antioxidants of natural origin have become the target of modern research [70].This study suggests that the extract and compound isolates of S. siamea exhibit promising antioxidant activities which align with previous reports.In a study by Oyebade et al. (2021) [71], the antioxidant properties of the leaf extracts of S. siamea were tested in vitro and the hexane, ethyl acetate, ethanol, and aqueous
In this work, the toxicity predictions of all the compound isolates were computed.The results revealed that all the compounds exhibited lethal dose values between 800 ˂ LD 50 ≤ 5000 mg/kg with toxicity classes 4 and 5 suggesting their slight toxicity and no compound favored acute toxicity [75].The prediction tool also showed toxicological (hepatotoxicity, carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity) endpoints.Accordingly, all the computed compounds revealed no hepatotoxicity results, thereby no problem with liver functions.All the compounds exhibited no cytotoxicity and except betulinic acid (4), all the compounds also revealed no carcinogenicity properties which were consistent with the prediction results of ciprofloxacin.Only chrysophanol (3) displayed mutagenicity properties and except glyceryl-1-hexacosanoate ( 5), all the compounds exhibited immunotoxicity properties (Table 4).Thus, the majority of the ADMET properties revealed that the investigated compounds possess drug-like properties and we suggest further experimental and computational studies.

Molecular docking studies
In this study, the bacterial protein targets were selected based on their metabolic importance to the microorganisms, the in vitro antibacterial and antioxidant profiles of the pathogens hosting them, and the likeness of the compounds to the co-crystalized ligands in the protein complex before preparation.DNA gyrase plays a vital role in the survival of bacterial cells and is mostly used as a drug target for in silico computational studies [77].
Reports on the DNA gyrase revealed that its enzymatic activities are critical in transcription, initiation, and elongation during DNA replication, DNA superhelicity regulation, and chromosome decatenation [78].Pyruvate kinase (PK), the enzyme at the last stage of glycolysis catalyzes the formation of adenosine triphosphate (ATP) and pyruvate by transferring a phosphoryl group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP) [79].PK plays a crucial role in metabolic flux distribution and energy generation.Inhibition of PK disrupts the metabolic stability and energy production of bacteria [80].. Streptococcal streptopain is also an important virulence factor leading to different streptococcal diseases including glomerulonephritis, fever, shock-like syndrome, and pharyngitis [81],.Thus, considering the aforementioned factors, we showed a desire to select the bacterial protein targets for this study.
The in silico molecular docking analyses of compounds exhibiting promising in vitro activities were performed and results showed that lupeol (1), β-sitosterol (2a), chrysophanol (3), and betulinic acid (4) displayed promising binding affinities against all the protein targets.The binding scores, hydrogen bonds, and residual amino acid interactions of the compound isolates along with reference drugs are depicted in Tables 5 and 6, and their 2D and 3D representations are presented in Figs. 5, 6, 7, 8 and 9. Chrysophanol (3) exhibited the highest binding affinity against all bacterial protein targets which were in line with the in vitro activities of the compound.During molecular docking studies, hydrogen bonds are among the most fundamental evidence that have to be considered while identifying active sites and they are essential bonds in determining the stability of the protein structure.This is because proteins are comprised of OH and NH groups which can donate electrons and hydrogen bonds to other groups.Therefore, hydrogen bonds help to specify protein-ligand interactions, thereby stabilizing the ligand in the binding site [82].Accordingly, chrysophanol (3) showed ≥ 2 H-bonds with all protein targets and thus exhibited better stability than the other compound isolates.
DNA gyrase B belongs to the bacterial type II A topoisomerase enzymes and regulates the topology of DNA during replication, recombination, and transcription by importing transient breaks to DNA strands and providing the necessary energy for catalytic functions [77].Lupeol (1), chrysophanol (3), and betulinic acid (4) showed minimum binding affinities of -6.6, -7.2, and − 7.0 kcal/mol, respectively, suggesting their inhibitory effects against E. coli DNA gyrase B. The binding scores of chrysophanol (3) and betulinic acid (4) were also comparable to the ciprofloxacin standard (-7.6 kcal/mol) and the scores were consistent with the in vitro activities of the compounds.The molecular docking interactions revealed a set of one H-bond (Glu-50) for lupeol (1) and three H-bonds each for chrysophanol (3) (Asn-46, Gly-77, and Pro-79) and betulinic acid (4) (Asn-46, Val-120, and Glu-50) of which the latter two compounds showed one common H-bond interaction (Asn-46) with ciprofloxacin (Arg-76, Asn-46, Asp-73).In addition, considerable residual amino acid interactions were observed for the compounds (Table 5; Fig. 5).
Nowadays, antibiotic resistance has become a key problem in various virulent pathogens due to the widespread usage of antimicrobial drugs [69].Many antibiotics now in application have negative side effects such as immunosuppression, hypersensitivity, and toxicity creating public health problems.Thus, the search for alternative therapies from plant-based natural products has become the concern of many research activities.Our research findings correlated the in vitro and in silico techniques to identify potential antibacterial and antioxidant compounds from the roots of S. siamea and the results showed notable progress compared to previous findings.Though the in vitro antibacterial and antioxidant investigations of the extract and isolated compounds from S. siamea aren't novel, we noticed that the previous studies lack further experimental and computational validations.In our case, the in vitro activities of the compound isolates were supported by in silico pharmacokinetic and molecular docking computations to verify possible therapeutic properties.Thus, we believe that the contributions of this work to the scientific world are vital and we suggest further investigations to study their efficacy in vivo.
In the present study, six known compounds were reported along with in vitro antibacterial, antioxidant, in silico ADMET properties, and molecular docking studies.The in vitro antibacterial evaluations revealed that the roots extract (18.00 ± 0.00 mm) and chrysophanol (3) (16.33 ± 0.24 mm) exhibited the highest inhibition diameters against E. coli, at 50 mg/mL and 2 mg/mL, respectively.The comparable IC 50 values of chrysophanol (3), betulinic acid (4), and β-sitosterol (2a) (IC 50 : 1.71, 2.06, and 2.35 µg/mL, respectively), to ascorbic acid (IC 50 : 0.53 µg/mL) were also evidence for the potential antioxidant potency of the compounds.The in silico pharmacokinetics, ADMET, and drug-likeness properties in combination with the molecular docking results of lupeol (1), β-sitosterol (2a), chrysophanol (3), and betulinic acid (4) suggest potential uses of these compounds as prospective antibacterial and antioxidants.Thus, the present study supports the traditional relevance of S. siamea, and further works are recommended on other biological activities of the plant.

Table 3
In silico drug-likeness and ADME properties of the isolated compounds (1-5) computed by the SwissADME online web tool