Antibacterial and Antioxidant Efficacies of Secondary Metabolites from the Roots of Cyphostemma adenocaule: A Combined In Vitro and In Silico Study

Cyphostemma adenocaule is a therapeutic plant traditionally used to treat rabies, snake bite, diarrhea, and wound healing. To address the bioactive compounds exhibiting these activities, we performed a comprehensive study on the roots of the plant. Thus, the present study aims to inspect the in vitro antioxidant and antibacterial efficacies of compounds isolated from the combined dichloromethane : methanol (1 : 1) and methanol extracts of C. adenocaule along with the in silico study of their interaction with selected protein targets. The silica gel column chromatography technique was used for the isolation of compounds, and the antibacterial and antioxidant activities were evaluated using agar disc diffusion and DPPH radical scavenging assays, respectively. Furthermore, in silico molecular docking screening, pharmacokinetics, and toxicity protocols of the compound isolates were performed to offer the potential applications of the compounds in developing novel medications. A BIOVIA Discovery Studio in combination with AutoDock Vina 4.2 software, SwissADME, and ProTox-II prediction web tools were used to generate the molecular docking, pharmacokinetics, and toxicity profiles, respectively. Notably, the chromatographic separation of the combined extracts yielded six known compounds, namely, β-sitosterol (1), 3-hydroxyisoagatholactone (2), ε-viniferin (3), myricetin (4), tricuspidatol A (5), and parthenocissin A (6). The in vitro antibacterial activities revealed the highest inhibition zone by tricuspidatol A (5) (16.67 ± 0.47), showcasing its potent activity against S. aureus at 2 mg/mL, compared to ciprofloxacin (21.50 ± 0.41). ε-Viniferin (3) (IC50: 0.32 μg/mL) exhibited greater antioxidant activity than the others and displayed promising results compared to ascorbic acid (0.075 μg/mL). The molecular docking study revealed the highest binding affinity by ε-viniferin (3) (−9.9 kcal/mol) against topoisomerase II α. 3-Hydroxyisoagatholactone (2) and ε-viniferin (3) fulfilled Lipinski's rule with no violation, and the organ toxicity predictions revealed that all the compounds showed no cytotoxicity and hepatotoxicity effects. Thus, this study's combined in vitro and in silico outcomes suggest the potential use of the isolated compounds in drug discovery and support the traditional relevance of C. adenocaule.


Introduction
Te biological activities and therapeutic values of medicinal plants rely on the existence of phytocompounds which generate a certain physiological response in the human body [1].Te most vital of these biologically active substances includes terpenoids, alkaloids, saponins, polyphenolic compounds, tannins, essential oils, and vitamins [2].Plenty of studies revealed that these bioactive compounds exhibit a broad array of biological properties such as anticancer, antioxidant, anti-infammatory, antifungal, antibacterial, antidiabetic, and antimalarial activities [3].
Te genus Cyphostemma (Vitaceae family) contains more than 250 taxonomically identifed species disseminated all over the tropics and subtropics of the world [4] with a wide spectrum of traditional uses to cure toothache, rabies, snake bite, tumor, infammation, malaria, marasmus, kwashiorkor in children, diarrhea, syphilis, and urinary and tract infections [5,6].Cyphostemma adenocaule (Steud.ex A. Rich.) (Figure 1) is one of the most prevalent vegetable plants grown in many parts of Africa, i.e., Ethiopia, Angola, Nigeria, Eritrea, Ghana, Uganda, Congo, Senegal, Malawi, and Mozambique [7,8].In Ethiopia, the plant has diferent vernacular names, such as Hareg Temen (Tigrinya), Aserkuh Aserkush (Amharic), and Hida Bofa (Afan Oromo) [9].In Tigray, Northern Ethiopia, the fresh roots of the plant have long been used against rabies, snake bite, and wound healing [10] and traditional healers around Gondar, Ethiopia, use the boiled root of C. adenocaule with milk and drink it to treat rabies [11].
Te phytochemistry of the plant revealed many natural substances including favonoids, stilbenes derivatives, coumarins, alkaloids, terpenoids, saponins, and tannins [12,13].Tese phytocompounds reveal diverse biological activities, including antifungal, antioxidant, antibacterial, chemopreventive, hepatoprotective, and antiproliferative activities [14,15].β-Sitosterol, betulin, betulinic acid, lupeol, cyphostemmic acids (A, B, C, and D), zizyberanal acid, and epigouanic acid A were among the reported terpenoid compounds in the stem barks and roots extract of C. adenocaule [13,16].Tus, motivated by its wide spectrum of traditional uses, the present study aims to inspect the in vitro antioxidant and antibacterial efcacies and in silico cytotoxic properties of compounds isolated from the combined CH 2 Cl 2 : MeOH (1 : 1) and methanol root extracts of C. adenocaule along with computational study of their interaction with selected protein targets.

Plant Material.
Te roots of C. adenocaule (wild climbing herb) were harvested from the mountains of Adama City, Ethiopia, in October 2021.After collection, the plant material was authenticated by Mr. Melaku Wendafrash (chief botanist).A voucher number of the specimen was designated by an accession number (HCA003) and deposited at the National Herbarium of Ethiopia, Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia.
Te plant samples were washed successively with tap and distilled water and subjected to air drying for three weeks at room temperature without direct exposure to sunlight.

General Experimental Procedures.
Te roots of C. adenocaule were fnely milled with a grinder (Shanghai Jingke, JK-HSG-100A, China) and extracted with CH 2 Cl 2 (98% Alpha Chemika, India) : MeOH (1 : 1) and MeOH (99.8%,Loba Chemie, India).Te solvents were freed using a rotary evaporator (DW-RE-3000, China) maintaining the temperature at 40 °C.Te crude extracts were chromatographed on silica gel (60-120 and 60-200 mesh, Merck, India) using column chromatography and eluted by dissolving in an appropriate solvent.For purity analysis, TLC aluminum plates (0.25 mm) coated with high-purity grade silica gel (230-400 mesh, pore size 60 Å, Merck Grade 64271, Darmstadt, Germany) were applied.Te fractions of compounds were visualized under a UV light (UV cabinet, UV4AC6/2, CBIO Bioscience and Technologies, Beijing, China, at 254 nm and 365 nm), spraying with 1% vanillinsulfuric acid reagent followed by direct heating over a stove at 110 °C.NMR spectral results were recorded in deuterated chloroform and methanol on 400 MHz Bruker AVANCE III NMR instruments.Te chemical shift (δ) values were conveyed in ppm and referred against the resonance values of the solvents at 3.31 ppm (δ H ) and 49.1 ppm (δ C ) for deuterated methanol and 7.28 ppm (δ H ) and 77.2 ppm (δ C ) for deuterated chloroform.Melting points were determined on a Japson Melting Point Apparatus (JA90161, India).Furthermore, the antioxidant properties of the plant extracts were achieved on a UV-Vis spectrophotometer (CE4001 UV/VIS, Cambridge, UK) equipped with tungsten and deuterium lamps.A hood with UV-radiation and laminar air fow, dimethyl sulfoxide, Muller-Hinton Agar (MHA) (Micro Express, India), sterilized needle, autoclave, incubator, micropipette (1000 μL), and Petri dishes (90 mm) were used for the antibacterial testing assays.All the solvents and reagents used were of analytical grade.

Extraction and Isolation.
Te pulverized roots of C. adenocaule (600 g) were successively extracted by a cold maceration technique with 3 L of CH 2 Cl 2 : MeOH (1 : 1) followed by an equal volume of methanol for 72 h each with continuous shaking over an orbital shaker (VRN-200, Gemmy industries, Taiwan) at room temperature.Te extracts were fltered over gravity with a Whatman No. 1 flter paper, and the fltrates were concentrated in vacuo on a rotary evaporator at 40 °C [17,18] to generate 22 and 28 g of CH 2 Cl 2 : MeOH (1 : 1) and methanol extracts, respectively.Both extracts exhibited similar TLC profles and were mixed to obtain 50 g of the crude extract.

In Vitro Antibacterial Activity
2.4.1.Bacterial Strains.Te isolated compounds were examined in vitro for their antibacterial efciency against four bacterial strains named Escherichia coli (ATCC-25922), Staphylococcus aureus (ATCC-25923), Pseudomonas aeruginosa (ATCC-27853), and Streptococcus pyogenes (ATCC-19615) which were selected based on their prevalence and common health problems in Ethiopia, availability, phenotype and genotype information, and relatedness to the ethnomedicinal relevance of the plant.Te bacterial pathogens were obtained from the Ethiopian Public Health Institute (EPHI), and the experimental tests were attained at the Research Laboratory of Microbiology, Adama Science and Technology University, Adama, Ethiopia.

Antibacterial Activity Assay.
Te in vitro antibacterial activities were adopted as per the previous experimental protocol [20] using DMSO and ciprofoxacin as negative and positive controls, respectively.Te entire tests were performed in triplicate, and the results were reported as mean ± SEM with the help of statistical software.

In Vitro Antioxidant Activity.
Te in vitro antioxidant activities were also adopted as per the previous experimental protocol [21] using ascorbic acid as positive control.Te absorbance value of a DPPH solution in methanol (negative control) was measured and the antioxidant power of each compound was described in terms of scavenging strength (%) using equation ( 1) [21].Te results were reported as mean ± SEM and the IC 50 values were computed from the relationship curve of plots of the concentration of samples against the antioxidant activity mean (%).

DPPH radical scavenging power
where A and A 0 indicate the absorbance of the sample solution in methanol + DPPH radical and DPPH radical in methanol, respectively.[22][23][24].
Water molecules and heteroatoms (phosphate, native ligands) were removed in the Discovery Studio Visualizer software; polar hydrogens were added, and proteins were saved in PDB format.Afterwards, Kollman charges and AD4-type atoms were added for the proper optimizations using MGL tools 1.5.7 and the proteins were saved in PDBQT format.Finally, the lowest energy conformations of the ligands were used as an input to run out the molecular docking simulations [25].Ten, the PDB format of the compounds was computed using MGL tools, Gasteiger charges were added, the torsion angle of the ligand was adjusted, and the proteins were saved in PDBQT format.Te active sites were established by studying the binding interaction of the receptor and the ligand and the molecular dockings were run on the active sites of the prepared target proteins.Te running process was achieved by building a grid box (50 × 50 × 54 Å) with a grid spacing of 0.375 Å and covering the active sites.Te docking process was achieved using the AutoDock Vina 4.2 program and all the fles saved in the working directory were run via the command prompt.For each ligand scored, nine diferent conformations were produced.Conformations of the least (stable) binding affnity and root mean square deviation (RMSD) were chosen for evaluating the interactions between the ligands and receptors.Finally, the docking analysis was computed using the BIOVIA Discovery Studio Visualizer 2021 [26].

Spectral and Statistical Data
Analyses.MestReNova software (version 14.2) was used for processing (apodization, Fourier transformation, and phase correction) of NMR raw data.Te antibacterial and antioxidant data were tabulated in a Microsoft Excel spreadsheet and the values were conveyed as mean ± standard error of the mean (SEM) (antibacterial) and % of scavenging activity (antioxidant).Te antibacterial activities were evaluated by comparing the inhibition zones of the compounds with the control drug (ciprofoxacin) and the solvent (DMSO).Te IC 50 scores of the compounds were computed from the relationship curves of the plots and compared to ascorbic acid.
Te spectral analyses of the compound were supported by 2D NMR correlations.Accordingly, the COSY spectrum exhibited 3   S2).Finally, the spectral data were in close agreement with the reported values of 3-hydroxyisoagatholactone (2) [33] (Figure 2).
Compound 6 was isolated as a brown amorphous solid using 90% EtOAc in n-hexane as eluent.Te 1   S6).
In its 13   13 C NMR spectral data of the compound (Supplementary materials, Table S6).

Antibacterial Activity.
Antimicrobial resistance has been recognized among the major threats in folk medicine and food-producing animals [40].Recent WHO reports also revealed alarming levels of antimicrobial resistance across many parts of the world [41].To overcome this challenge, we conducted a comprehensive study on four bacterial strains (E.coli, S. aureus, P. aeruginosa, and S. pyogenes) to support the traditional relevance of the roots of C. adenocaule and introduce alternative sources of medicine.Te prevalence of bacterial diseases in Ethiopia and traditional practices of the plant against them motivated us for the selection of the microorganisms.
In the present work, the in vitro antibacterial efcacy of the CH 2 Cl 2 : MeOH (1 : 1) extract and the isolated compounds (1-6) of C. adenocaule were evaluated against the four bacterial pathogens at four diferent concentrations (extract: 50, 25, 12.5, and 6.25 mg/mL and isolated compounds: 2, 1, 0.5, and 0.25 mg/mL).For the extract, the highest inhibitory diameters were recorded against E. coli (18.00 ± 0.00 mm) and S. aureus (17.16 ± 0.24 mm) at 50 mg/ mL, compared to ciprofoxacin which showed 21.33 ± 0.47 and 21.50 ± 0.41 mm for E. coli and S. aureus, respectively, whereas, at the smallest concentration (6.25 mg/mL), the extract also displayed a better inhibition zone against E. coli (9.83 ± 0.24 mm) (Table 1).Our fndings corroborate earlier research studies wherein the chloroform (22.5 mm) and methanol (15.0 mm) root extracts of C. adenocaule exhibited comparable antibacterial activities against E. coli and promising zones of diameters were also observed for chloroform (15.0 mm) and methanol (10.5 mm) extracts against S. aureus, at unknown concentrations [42].In the present work, the activity of the extract was also compared to the antibacterial susceptibility of C. adenocaule extracts included in the survey of antimicrobial medicinal plants from Uganda [43].Again, better antibacterial activities were recorded from our fndings compared to the report of similar bacterial strains.Based on the data presented, the study established that the CH 2 Cl 2 : MeOH (1 : 1) root extract of the plant exhibits antibacterial properties against the chosen bacterial strains, which support its traditional relevance.
Te antibacterial properties of all the compounds were compared to similar reports of the plant [33].Accordingly, β-sitosterol (1), 3-hydroxyisoagatholactone (2), ε-viniferin (3), and tricuspidatol A (5) were the subjects of the previous report, and comparable antibacterial activities were reported with the present study.Te report revealed the highest inhibitory diameters for ε-viniferin (3) (12.22 ± 0.74), β-sitosterol (1) (10.90 ± 1.54), and tricuspidatol A (5) (10.79 ± 0.24) against S. aureus at 1 mg/mL compared to chloramphenicol standard (11.76 ± 0.77).Regardless of the reference drug, the results were as good as compared with the fndings of the present study (Table 1).Te previous report also suggested that 3-hydroxy-isoagatholactone (2) was identifed and tested for the frst time and displayed low Journal of Tropical Medicine to moderate antibacterial activities, whereas, in the present work, the activities of the compound were also studied and better results were generated.Many works have been reported on the antimicrobial activities of β-sitosterol (1) and results revealed that the compound possesses low to moderate activities against diferent bacterial pathogens [31,44,45].Te previous activities were also in good agreement with the fndings of the present study.Tus, the in vitro antibacterial experimental results support the use of C. adenocaule in traditional medicinal systems.
Te IC 50 values were calculated from the logarithmic regressions of the relationship curves on a Microsoft Excel 2016 spreadsheet.Accordingly, the trend line analyses of the compounds generated the following equations, and the values were calculated using logarithmic relationships (Figure 4).β-Sitosterol (1)

Molecular Docking Studies.
In the present study, the protein targets were selected based on their metabolic importance to the microorganisms, the respective strains' in vitro antibacterial and antioxidant activities, and the similarity of the compound to the cocrystalized ligands in the protein complex.In the PDB database, proteins are found in complexes with ligands (cocrystallization).Target proteins are prepared from the cocrystallized structures by removing water molecules, identifying the appropriate binding sites, and adding polar hydrogens.Te prepared proteins must have a suitable binding pocket/active site into which a drug or drug-like molecule can bind, and the structures of the cocrystallized ligands play prominent  factors in determining similarities with the isolated secondary metabolite candidates for docking.Most of the protein complexes in the PDB database are made up of multiple ligands and active sites.Tus, a cocrystallized ligand having related structural features with our candidate compounds suggests that the active site of that specifc protein is a promising candidate to be considered for molecular docking study, and hence, the respective proteins are given prior consideration.

Molecular Docking Study against Topoisomerase II α.
Notably, the uses of C. adenocaule against various types of cancer cells have been reported.Matata and co-workers reported that the root extracts of the plant exhibit cytotoxic properties against HeLa cervical cancer cell lines [46].According to the report, the ethyl acetate fractions showed high toxicity activities against the HeLa cervical cancer cells with IC 50 � 3.4 ± 0.  9).were verifed by myricetin (4) against CYP1A2, 3-hydroxyisoagatholactone (2), and ε-viniferin (3) against CYP2C9 and myricetin (4), and tricuspidatol A (5) against CYP3A4.
Te toxicological classifcations of drugs/chemicals are convoyed in terms of LD 50 (median lethal dose) which is the amount of drug that is lethal to one-half (50%) of the experimental organisms viable to it [48].Acute toxicity studies  are performed to have safer routes of exposure (i.e., inhalation, dermal, and oral) and rodents are mostly employed to evaluate the lethal dose [49].According to EPA's 4 categories of hazard classifcations, compounds/chemicals with LD 50 ≤ 50 mg/kg are highly toxic, 50 < LD 50 ≤ 500 mg/kg (moderately toxic), 500 < LD 50 ≤ 5000 mg/kg (slightly toxic) and LD 50 > 5000 mg/kg (safe chemicals) [49].Hence, all the investigated compounds showed no acute toxicity (Table 10).Te prediction outcomes also revealed that all the compounds showed no cytotoxicity and hepatotoxicity properties.Tis was consistent with the toxicity prediction values of ciprofoxacin.ε-Viniferin (3) and myricetin (4) were active towards carcinogenicity, β-sitosterol (1), 3-hydroxy isoagatholactone (2), ε-viniferin (3), and parthenocissin A (6) showed immunotoxicity, and only myricetin (4) was active towards mutagenicity.Tus, based on the prediction methods, the compounds of study possess drug candidate properties.

Conclusion
Te emergence of novel pathogens and the growth of antibiotic resistance raise momentous apprehensions in the healthcare industry.In this study, six compounds were isolated from C. adenocaule (roots) and their in vitro and in silico antioxidant and antibacterial properties were studied.Te antibacterial tests showed that the compounds exhibit valuable activities, and at the smallest concentration (0.25 mg/mL), the highest inhibition zones were displayed by tricuspidatol A (5) (9.83 ± 0.24) and parthenocissin A (6) (9.67 ± 0.47) against E. coli, which were smaller than the reference drug (ciprofoxacin) but unquestionably significant.Te antioxidant efcacy of the compounds was also promising.ε-Viniferin (3) (IC 50 � 0.32 μg/mL) exhibited the strongest DPPH radical scavenging activity compared to the others.Te binding afnities of the compounds were  also consistent with the in vitro experimental studies.ε-Viniferin (3) (−9.9 kcal/mol) displayed the highest binding afnity against topoisomerase II α protein target.Te toxicity predictions showed no cytotoxicity and hepatotoxicity efects for all the compounds consolidating their drug likeness properties.Tus, the CH 2 Cl 2 : MeOH (1 : 1) extract of C. adenocaule is suggested as a valuable source of natural antibacterial and antioxidant compounds, contributing opportunities for emerging novel pharmaceuticals, which also supports the traditional uses of C. adenocaule.We also suggest further eforts to attempt the plant's phytochemistry to isolate novel compounds with additional biological activities that are not included in this study.

Table 6 :
Molecular docking study of the isolated compounds (3-6) and ascorbic acid against human myeloperoxidase.

Table 8 :
In silico drug-likeness properties of the isolated compounds (1-6) generated by SwissADME.MF, molecular formula; MW, molecular weight; NHA, number of hydrogen acceptors; NHD, number of hydrogen donors; NRB, number of rotatable bonds; TPSA, total polar surface area.