In Vitro Antibacterial, DPPH Radical Scavenging Activities, and In Silico Molecular Modeling of Isolated Compounds from the Roots of Clematis hirsuta

Clematis hirsuta is one of the traditional medicinal plants used in Ethiopia to treat different ailments, such as cancer and diseases related to the respiratory system. This study aimed to isolate the phytochemical components of the root of C. hirsuta and evaluate their in vitro and in silico biological activities. Oleic acid (1), palmitic acid (2), sterols (3 and 4), boehmenan (5), and carolignans E (6 and 7) were isolated by silica gel column chromatography and preparative thin layer chromatography and characterized by NMR spectroscopy. Compounds 5–7 were isolated from the plant for the first time. At 5 mg/mL, the inhibition zone of evaluated compounds ranged from 8.80 to 11.10 mm against all selected bacteria. The MIC of the MeOH and n-hexane: EtOAc (1 : 1) extracts was greater than or equal to 50 mg/mL against all selected bacteria. At 62.5 μg/mL, the % DPPH radical scavenging activity of tested compounds ranged from 30.3% to 92.1% with an IC50 value of 19.4 to 2.1 μg/mL. The results of molecular docking studies indicated that the docking scores of compounds 3–7 ranged from −6.4 to −7.9 kcal/mol against E. coli DNA gyrase B, −8.3 to −9.0 kcal/mol against the Pseudomonas quinolone signal A, −7.1 to −8.5 kcal/mol against pyruvate kinase M2, and −7.9 to −8.5 kcal/mol against human topoisomerase IIβ. The results of the in silico antibacterial activity of compounds 3, 5, and 6 supported the in vitro antibacterial test results. Compound 5 had a better docking score against human topoisomerase IIβ than the other test samples demonstrating its potential as an anticancer agent. Therefore, compounds 3–7 could be considered as a lead for developing antibacterial and anticancer drugs. Moreover, the presence of these active phytochemicals supports the traditional use of this plant against cancer and bacteria.


Introduction
Clematis hirsuta Perro & Guill (Ranunculaceae) is a shrub that belongs to the genus Clematis [1].Te plant is endemic to Cape Verde, the Sahara, tropical Africa, and the South West Arabian Peninsula.It can grow up to 4 m and contains leaves of the pinnate type with 5 leafets.Te shape of the leaves is suborbicular to ovate with tips of acuminate, acute, or subobtuse with cordate to rounded and rarely truncate at the base.Te margins of the leaves are longer in the central lobe and shorter on each side.Te plant contains inforescences composed of many fowers with pedicels 1 to 3 cm long and 1 to 2.7 cm long cream or white sepals.It has a fower bud with a spherical to ellipsoid shape containing rounded to acuminate tips [1].
C. hirsuta, locally called Hidda feetii in Afan Oromo [2], is a plant that has traditionally been used to treat diferent diseases including swelling, cough, cataracts, and leishmaniasis [3][4][5][6].Te fresh leaves of the plant are also used to treat earache, cancer, and tuberculosis [7,8].Its roots are administered orally to treat respiratory tract problems [5].Te whole part of the plant is used for the treatment of fungal and trypanosomal diseases [9] and wounds [10].Pharmacological reporting has shown that the various solvent extracts of the leaves and roots of C. hirsuta exhibit antibacterial activities [2,[11][12][13].Te MeOH extracts of the root, the petroleum ether, CHCl 3 , and EtOH extracts of the leaves were also reported to have DPPH radical scavenging activities [2,13].Te phytochemical screening of the root of the plant revealed the presence of alkaloids, saponins, tannins, favonoids, phenols, and glycosides [2].Te aerial part of C. hirsuta was also reported to have β-amyrin, lupeol, β-sitosterol, oleanolic acid, and stigmasterol glycoside [14].
Despite the tremendous traditional use of the plant against various life-threatening diseases, there is no prior report on the isolation and characterization of secondary metabolites from the root extracts of C. hirsuta.Furthermore, the biological activities of the root of the plant were also not well explored.Terefore, this work aimed to isolate and characterize phytochemicals from the root extracts of C. hirsuta and assess their antibacterial and 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging activities.Hence, in this paper, we presented the fndings on the phytochemical constituents, in vitro antibacterial, and DPPH radical scavenging activities of root extracts of C. hirsute for the frst time.Te results of the in silico molecular docking analysis, drug-likeness, and toxicity of isolated compounds were also included.

Plant Material Collection, Identifcation, and Preparation.
Te root of C. hirsuta was collected in July (Summer 2021) from Waldoro village, Homa Kebele, Abbay Chomman Woreda, Horo Guduru Zone, Oromia Regional State, which is about 400 km west of the capital, Addis Ababa, Ethiopia.Te plant was identifed with the help of available literature and authenticated (TD05/2021) by botanist Mr. Melaku Wendaferash at the National Herbarium, Department of Biology, and Addis Ababa University, Ethiopia.Ten, the collected plant material was immediately brought to the Organic Chemistry Laboratory of Adama Science and Technology University and allowed to dry in the open air at room temperature under shade.Te dried plant material was then pulverized using an electric grinder and stored at 4 °C.
Fractions 28-43 (230 mg), which were eluted using nhexane: EtOAc (7 : 3), were further fractionated with the same eluent using silica gel column chromatography (size: 60 mm internal diameter by 360 mm length with 14/23 joint type) to obtain compound 1 (15 mg).Likewise, fractions 44-46 (330 mg), which were eluted with n-hexane: EtOAc (7 : 3), were eluted in isocratic mode using column chromatography with silica gel to obtain compound 2 (16 mg).Fractions 47-55 (165 mg), which were eluted using n-hexane: EtOAc (65 : 35), were fractionated in isocratic mode on silica gel column chromatography to obtain a mixture of compounds 3 and 4 (32 mg).Fractions 86-95 (330 mg) were eluted using n-hexane: EtOAc (1 : 9).Using the same solvent ratio, the fractions were eluted in isocratic mode on silica gel column chromatography and further purifed using PTLC to obtain compound 5 (18 mg) and a mixture of compounds 6 and 7 (30 mg).2.3.1. Disk Difusion Method.Te evaluation of the antibacterial activity of extracts and isolated compounds was carried out using the disk difusion method against clinical isolate bacteria, including two Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes ATCC 19615) and two Gram-negative bacteria (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) using the standard procedure [15].Tus, 38 g of Mueller-Hinton agar medium (MHA) was dissolved in 1 L of distilled water and heated to dissolve until completely and allowed to cool.After sterilization using an autoclave at 121 °C for 15 minutes, 25 mL of the medium was dispensed into a 90 mm Petri dish to a depth of approximately 4 mm and allowed to solidify at room temperature and stored at 4 °C.Te test bacteria was diluted in sterile Muller-Hinton broth to adjust the turbidity to the 0.5 McFarland BaCl 2 standard solution to get 1.5 × 10 8 cfu/mL bacteria suspension.Within 15 min, each inoculum was inoculated in a Petri dish using a sterile swab.A 6 mm sterile disc made of Whatman flter paper No. 1 was saturated with 20 μL of 100 and 50 mg/mL of each extract and with 5 and 2.5 mg/mL of each isolated compound and then placed on the surface of the Petri dish.Te Petri dish was incubated at 37 °C for 24 hours.Te diameter (in millimeters) of the inhibition zone was measured.Te extent of activity of the samples was compared to a sterile 6 mm disc saturated with 20 μL (10 μg) of 0.5 mg/mL ciprofoxacin.All analyses were carried out in triplicate.

Minimum Inhibitory Concentration (MIC) of Extracts.
Te agar dilution method was used to evaluate the MIC of the MeOH and n-hexane: EtOAc (1 : 1) extracts using the procedure [16,17] with some modifcations against the selected bacteria.In detail, the MHA media was prepared as described above.A stock solution of 100 mg/mL was prepared by dissolving 400 mg of each extract in a small amount 2 Advances in Pharmacological and Pharmaceutical Sciences (2 mL) of DMSO.Afterwards, it was adjusted to the fnal volume of 4 mL using the MHA solution and serially diluted to the working solution 50, 25, 12.5, 6.25, and 3.125 mg/mL in the MHA solution.About 500 μL of each concentration was distributed into 100 mL labeled sterilized beakers and mixed with 24.5 mL of MHA media solution.Te mixtures were then poured into a Petri dish labeled with the respective concentration of extracts.Following that the plate was allowed to dry at room temperature.Approximately 5 μL of suspension (density adjusted to 0.5 McFarland turbidity units, 1 to 2 × 10 8 cfu/mL) was transferred as a spot on each plate.Te plates were incubated at 37 °C for 24 hours.Te lowest concentration that completely inhibits visible growth, as judged by the naked eye, was taken as MIC.Agar plates without test extract and with standard antibiotics, ciprofoxacin (0.0625, 0.03125, and 0.015625 mg/mL), have been used as a negative and positive control, respectively.

Radical Scavenging Activity.
Te radical scavenging activity of the extract and isolated compounds was evaluated using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical according to the reported procedure [18].In detail, 4 mg of DPPH was dissolved in 100 mL methanol to obtain 40 μg/mL and stored in a dark bottle at 4 °C.Te crude extracts and isolated compounds were separately dissolved in methanol to prepare 1000 μg/mL.Using a two-fold serial dilution in methanol, 500, 250, 125, and 62.5 μg/mL working solutions were prepared.Ten, 1 mL of each was mixed with 1 mL of DPPH.After 30 min of incubation in darkness at room temperature, the absorbance was taken using a UV-vis spectrometer at 517 nm adding methanol as a blank.Te percent (%) of DPPH inhibited by the samples was calculated as (A c − A S /A c ) × 100%, where A c is the absorbance of the control and A s is the absorbance of the sample.IC 50 was calculated from the regression line [19].Ascorbic acid was used as standard.All analysis was carried out in triplicate.

In Silico Molecular Docking Analysis of the Isolated
Compounds.Te docking was conducted using the reported procedure [20].Specifcally, the structures of the compounds were drawn using ChemDraw 22.0 and saved in (.mol fle) format before they were optimized using Discovery Studio Visualizer 21.1 software [21] and saved in (.pdb fle) format.[21], the binding sites were identifed, and all ligands and water molecules were removed from the target proteins.Te docking was carried out using AutoDock Vina with MGL tools 1.5.6 and Python 3.10.9as supporting software.Each run was carried out for nine conformers.Te least binding energy conformer with the lowest root mean square deviation (RMSD) was selected and analyzed using the Discovery Studio Visualizer to show the interaction of the ligand and target enzymes in the form of a 2D and 3D structure.Te result was compared with the result of the standard antibacterial drug, ciprofoxacin, and the standard anticancer drug, abiraterone.

In Silico Pharmacokinetics and Toxicity of Isolated
Compounds.Te canonical simplifed molecular input line entry system (SMILES) was taken from the PubChem database and submitted to the SwissADME online tool to estimate physicochemical (molecular weight, number of rotatable bonds, number of hydrogen bond donors, number of hydrogen bond acceptors, and total polar surface area), lipophilicity (log P O/W ), and drug-likeness (Lipinski's rule) [22].For the prediction of in silico acute oral toxicity, organ toxicity (hepatotoxicity), and end points of toxicity (carcinogenicity, immunotoxicity, mutagenicity, and cytotoxicity), isolated compound SMILES was submitted to the Pro Tox II online tool [23].Te result of the isolated compounds was compared with those of ciprofoxacin and abiraterone.

Results and Discussion
3.1.Characterization.Seven compounds (Figure 1) including oleic acid (1), palmitic acid (2), sterols (3 and 4), boehmenan ( 5), and carolignans E (6 and 7) were isolated and characterized from the root extract of C. hirsuta.Te structures of the isolated compounds were elucidated using NMR spectroscopy with the details presented as follows.
Compound 6 was obtained as a yellowish jelly showing the TLC spot at R f value 0.5 with n-hexane: EtOAc: CHCl 3 : MeOH (5 : 3 : 1 : 1) as a mobile phase.Te NMR spectral data revealed that the sample is a mixture of compound 6 (major) and compound 7 (minor) (Figures S13-S15).Te complete assignment of the NMR spectral data is given in Tables 2 and  3. Te two compounds are almost similar in their spectral data except signal due to H-7′ which was observed at δ 4.92 and 4.94 ppm assigned to compound 6 and compound 7, respectively.Literature reports showed that the stereochemistry of these compounds at H-7 is diferent [28].In this regard, H-7′ appeared as a singlet at δ 4.92 (equatorial with H-8′) in compound 6; however, it appears as a doublet (J � 8.1 Hz) at δ 4.94 (axial with H-8′) in compound 7.
However, in the current report, due to the complexity of the 1 H-NMR spectrum, it was difcult to identify the multiplicity of H-7′ for compounds 6 and 7. Using the chemical shift diference of H-7′ in compounds 6 and 7 (Figure S13), the area under integration of H-7′ was determined.Te result indicated that the compositions of compounds 6 and 7 in the mixture were calculated as 57% and 43%, respectively.From Ochroma lagopus Swartz and Euphorbia sikkimensis plants, similar NMR spectral data of compounds 6 and 7 were reported for the compounds erythro-carolignan E and threo-carolignan E, respectively [27,29].Hence, the NMR spectral data of compounds 6 and 7 were compared (Tables 2 and 3) with the same compounds reported in the literature and found in good agreement [28].3.2.1. Disk Difusion Method.Te results of an in vitro antibacterial assay performed using a disk difusion method against clinical isolate bacteria including S. aureus, S. pyogenes, E. coli, and P. aeruginosa are shown in Table 4.As indicated, at 100 mg/mL, the MeOH extract showed a better inhibition zone with a diameter of 12.30 ± 0.21 mm against S. pyogenes.It showed the lowest inhibition zone with a diameter of 8.20 ± 0.12 mm against S. aureus.On the contrary, the extract of n-hexane: EtOAc (1 : 1) demonstrated the maximum inhibition zone with a diameter of 15.20 ± 0.17 mm against P. aeruginosa and showed the least inhibition zone (8.40 ± 0.26) against E. coli.

Antibacterial Activities
In order to fnd out the active constituents, the isolated compounds were also assessed for their antibacterial activity.Among the isolated compounds, compound 3 had an inhibition zone of 11.10 ± 0.17, 10.50 ± 0.21, 10.30 ± 0.24, and 10.60 ± 0.55 against S. aureus, S. pyogenes, E. coli, and P. aeruginosa, respectively.Te result turned out to be close to the activity displayed by ciprofoxacin.Compound 6 showed a comparatively better inhibition zone with a diameter of 10.40 ± 029 mm against E. coli.Te activity displayed by the root extract of C. hirsuta in the present study was comparable with antibacterial activity reported in the literature carried out on the CHCl 3 and MeOH extracts of C. hirsuta leaves where the CHCl 3 extract showed an inhibition zone of 12.33, 10.70, and 10.26 mm and the MeOH extract showed an inhibition zone of 8.50, 9.30, and 8.00 mm against P. aeruginosa, E. coli, and S. aureus, respectively [11].Tis confrms that the n-hexane: EtOAc (1 : 1) extract of the plant contains bioactive substances that have better activities against P. aeruginosa than against the other test bacteria.

Minimum Inhibitory Concentration (MIC) of Extracts.
Te results of the MIC assay of the plant root extracts performed using the agar dilution method against two Gram-positive clinical isolates (S. aureus and S. pyogenes) and two Gram-negative clinical isolates (E. coli and P. aeruginosa) are shown in Table 5.As indicated, the MIC results were identifed to be greater than or equal to 50 mg/ mL for both MeOH and n-hexane: EtOAc (1 : 1) extracts against the four bacterial strains.Unlike the MIC of the standard drug, ciprofoxacin was determined to be less than 15.625 μg/mL against all selected bacteria.Comparable results were observed from the MIC evaluation conducted on the MeOH and CHCl 3 extracts of the plant leaves against S. aureus, E. coli, and P. aeruginosa wherein the maximum Advances in Pharmacological and Pharmaceutical Sciences MIC value was found to be 60 mg/mL and the minimum MIC value was 30 mg/mL [12].

Radical Scavenging Activities.
Te result of the ability to inhibit the DPPH radical of extract and isolated compounds is presented in Table 6.At 62.5 μg/mL, the percent of DPPH radical inhibited using extracts of methanol and n-hexane: EtOAc (1 : 1) was 56.7% and 41.7%, respectively.Compounds 3, 5, and 6 inhibited 30.3, 92.1, and 86.5% of the DPPH radical, respectively.Compound 5 showed potent DPPH radical scavenging activity with an IC 50 value of 2.1 μg/mL which is comparable with ascorbic acid with an IC 50 value of 0.5 μg/mL.Te IC 50 values of compounds 3 and 6 were 19.4 μg/mL and 7.4 μg/mL, respectively.Compounds 5 and 6 showed higher radical scavenging activities than the extracts and other isolated compounds at 62.5 μg/mL, compared to the radical scavenging ability of ascorbic acid.Te strong DPPH radical scavenging activities of compounds 5 and 6 could be related to their number of hydroxyl groups which implies that phenolic compounds exhibit potent DPPH radical scavenging activities [30].It was found that the radical scavenging activity displayed by MeOH extract of the root of the plant was superior compared with literature reported for the same plant [2].

In Silico Molecular Docking Analysis of the Isolated
Compounds.Te molecular docking study of isolated compounds was carried out against targets, E. coli DNA gyrase B (PDB ID: 7P2M) and the Pseudomonas quinolone signal A, PqsA (PDB ID: 5OE4), to evaluate in silico antibacterial activities and against Pyruvate kinase M2, PKM2 (PDB ID: 4G1N), and human topoisomerase IIβ (PDB ID: 3QX3) to study the in silico anticancer activities of isolated compounds.
Te results of docking analysis against E. coli DNA gyrase B (PDB ID: 7P2M) and Pseudomonas quinolone signal A, PqsA (PDB ID: 5OE4) were compared with the standard antimicrobial, ciprofoxacin (Table 7).
Te docking study with 7P2M revealed that the binding afnity of the isolated compounds ranged from −7.9 to −6.4 kcal/mol compared to the binding afnity of ciprofoxacin (−7.4 kcal/mol).Te highest docking score was exhibited by compound 7 (−7.9kcal/mol) while the lowest was exhibited by compound 5 (−6.4 kcal/mol).As indicated, Te docking analysis against 5OE4 indicated that the binding afnity of isolated compounds ranged from −9.0 to −8.3 kcal/mol compared to the docking score of ciprofoxacin (−7.2 kcal/mol).Te highest binding afnity was exhibited by compound 3 (−9.0kcal/mol), while the lowest binding afnity was exhibited by compound 5 (−8.3 kcal/ mol).Compound 3 showed the highest docking score (−9.0 kcal/mol) without hydrogen bonding (Figure 2), while compound 5 showed hydrogen bonding through  amino acid residues which interacted with the lowest docking score (−6.4 kcal/mol).All the tested compounds showed greater afnity for PqsA compared to that of ciprofoxacin.In general, the in silico antibacterial study indicated that compound 3 comparatively showed good antibacterial activity compared to the rest of the isolated compounds.Terefore, the 2D and 3D interaction of  Advances in Pharmacological and Pharmaceutical Sciences compound 3 with PqsA is selectively compared to the 2D and 3D interaction of ciprofoxacin (Figure 2).Unlike compound 3, ciprofoxacin interacted with PqsA using hydrogen bonding through Arg-200 amino acid residues in addition to hydrophobic, electrostatic, and van der Waals interaction.Te 2D and 3D interaction of compounds 3-7 with 7P2M and 5OE4 is shown in Figures S16-S17.
Te results of the in silico antibacterial studies support the in vitro antibacterial activities, and therefore, the isolated compounds can be considered as potential antibacterial agents.Furthermore, it is noted that ciprofoxacin targets both DNA gyrase (topoisomerase II) and topoisomerase IV [31].Terefore, the inconsistency of the result for ciprofoxacin observed in in vitro with the in silico antibacterial activity test might be related to the mechanism of action of ciprofoxacin, as the in silico antibacterial activity evaluation was performed against a specifc enzyme.Tus, it can be concluded that the correlation between the number of hydrogen bonds and the values of the docking score is not proportional.As observed in this report, a docking result containing more hydrogen bonding can be found with a low binding afnity.For example, the results reported for molecular docking studies conducted against Plasmodium falciparum hexose transporter 1 (PfHT1) protein demonstrated that the ligand-protein complex with the highest number of hydrogen bonding was found with the lowest docking score [32].
Te result of docking analysis against pyruvate kinase M2, PKM2 (PDB ID: 4G1N), and human topoisomerase IIβ (PDB ID: 3QX3) was compared with the result of the standard anticancer drug, abiraterone (Table 8).
In conclusion, from in silico anticancer activity studies, compound 5 showed good activities against both cancercausing enzymes, 4G1N and 3QX3, and hence, selectively the 2D and 3D interaction of compound 5 with 4G1N is compared with the 2D and 3D interaction of abiraterone (Figure 3).For the rest of the isolated compounds, the 2D and 3D interactions with 4G1N and 3QX3 are shown in Figures S18 and S19, respectively.12 Advances in Pharmacological and Pharmaceutical Sciences 3.5.In Silico Pharmacokinetics and Toxicity of the Isolated Compounds.Table 9 shows the results of the drug-like properties calculated from the isolated compounds using SwissADME according to Lipinski's rule.As indicated, compounds 3 and 4 violate Lipinski's rule similar to the standard anticancer drug, abiraterone.However, compounds 5 and 6 showed two violations of Lipinski's rule implying that they cannot be administered orally as a drug [22].From Pro-Tox II analysis, acute oral toxicity, organ toxicity, and toxicological endpoints were predicted.Compounds 3, 4, 5, and 6 showed predicted class 4 toxicity and have shown immunotoxicity.In the same way, the acute oral toxicity prediction result for standard antibiotics, ciprofoxacin, indicated predicted toxicity class 4 and has shown only mutagenicity.Also, the standard anticancer drug, abiraterone, has shown a class 4 prediction of toxicity with the property of immunotoxicity.Terefore, none of the isolated compounds was shown to have acute toxicity due to the predicted LD 50 which is greater than fve [23].

Conclusion
In this study, seven compounds were identifed from the roots of C. hirsuta.Compounds 5-7 were reported for the frst time from the plant and the genus of the plant.For the secondary metabolites, the in vitro antibacterial activities conducted using the disk difusion method against four bacteria were validated by using in silico molecular docking analysis.Compound 3 exhibited relatively good antibacterial activity against S. aureus and P. aeruginosa bacteria, while compound 6 showed better activity against E. coli.Te MIC value of the extracts was greater than or equal to 50 μg/mL against all bacteria.Te extract of n-hexane: EtOAc (1 : 1) from the root of C. hirsuta contains phytochemical components which are the most potent antibacterial agents against Gram-negative bacteria, P. aeruginosa.Terefore, a high yield of the n-hexane: EtOAc (1 : 1) extract must be further studied to isolate a potent antibacterial agent against Gram-negative bacteria.On the contrary, even though two of Lipinski's rules of fve were violated, compounds 5 and 6 were considered strong DPPH radical scavengers, and therefore, compounds 5 and 6 should be modifed according to Lipinski's rules of fve so that they could be used as lead compounds in the development of anticancer drugs.Furthermore, phytochemicals isolated from the root of the plant and which showed in vitro antibacterial activities and in silico anticancer activities are substantial evidence for the traditional uses of the plant to treat diseases caused by bacteria and cancer-related diseases.In addition, we recommend toxicity and in vivo assays for compounds 5-7 that were isolated from the root of C. hirsute to evaluate their potential as antibacterial and anticancer agents.

Figure 1 :
Figure 1: Chemical structures of isolated compounds from the roots of C. hirsuta.
3, 6, and 7 can be considered as good antibacterial activity against E. coli.

Table 4 :
Bacterial growth inhibition zone of extracts and isolated compounds.

Table 6 :
DPPH radical scavenging activity of the extracts and isolated compounds.

Table 9 :
Drug-likeness predictions of isolated compounds computed by SwissADME.Note.MWT: molecular weight; NRB: number of rotatable bonds; NHA: number of hydrogen acceptors; NHD: number of hydrogen donors; TPSA: total polar surface area.