Interspecies Anticancer and Antimicrobial Activities of Genus Solanum and Estimation of Rutin by Validated UPLC-PDA Method

Solanaceae is one of the highly diverse plant families of which Solanum is the largest genera (1700 species) containing several pharmacological properties like anticancer and antimicrobial. This motivated us to explore the anticancer (against HepG2, HEK-293, and MCF-7 cells) and antimicrobial (against S. aureus, E. coli, P. aeruginosa, and C. albicans) properties of S. schimperianum, S. villosum, S. coagulans, S. glabratum, S. incanum, and S. nigrum along with rutin estimation by UPLC-PDA method. Of the studied Solanum extracts, S. nigrum exhibited significant cytotoxic property against HepG2 (IC50: 20.4 μg/mL) and MCF-7 (IC50: 30.1 μg/mL); S. coagulans showed toxicity against HepG2 (IC50: 28.4 μg/mL) and HEK-293 cells (IC50: 25.7 μg/mL) compared to 5-Fluorouracil (standard). Compared to these, extracts of S. coagulans and S. glabratum exhibited relatively high antimicrobial potency (MIC: 0.4-1.6 mg/mL). Nonetheless, all Solanum extracts significantly reduced the biofilm against PAO1-strain. Rutin was detected in all extracts with the highest content (53.79 μg/mg) in S. coagulans that supported its strong antimicrobial and anticancer properties. Molecular docking analysis showing strong binding of rutin with human DNA and proteins (DNA Topoisomerase IIα and E. coli DNA gyrase B) supported the anticancer and antimicrobial activities of Solanum species.


Introduction
Solanaceae is one of the highly diverse plant families and consisted of wide range of therapeutically important chemical entities. It consists of perennial trees as well as herbaceous annual species, widely distributed from deserts to rainforests. Of this, Solanum is considered as the largest genera with nearly 1700 species. Sixteen species of Solanum are reported to be found in the West and Southwest areas of Saudi Arabia [1]. The extracts of S. schimperianum are reported to possess antioxidant, antitrypanosomal [2], and antimicrobial properties [3]. S. villosum Miller extract showed strong in vitro cytotoxic property against breast cancer cells [4]. Also, proteins extracted from S. villosum leaves exhibited larvicidal properties against Anopheles stephensi, Culex quinquefasciatus, and Stegomyia aegypti and antimicrobial activities [5]. S. glabratum Dunal extracts possessed excellent activity against intracellular amastigotes of Leishmania infantum [6] and strong cytotoxic property against breast (MCF-7), hepatocellular carcinoma (HepG2), and cervix (HeLa) cancer cells [7]. S. nigrum Linn extract exhibited strong cytotoxic property against human malignant melanoma cell line (A-375) and breast cancer cells by [8]. S. nigrum seed extracts exhibited moderate antiviral activity against hepatitis C virus NS3 protease [9]. Phytochemical investigation of S. schimperianum Hochst revealed the presence of a variety of steroidal, terpenoidal, and flavonoidal compounds like lupeol, -sitosterol, -sitosterol glucoside, oleanolic acid, ferutinin, 5-hydroxy-3,7,4'-trimethoxyflavone, retusin, and kaempferol-3-O--d-glucopyranoside [10] while S. incanum L. was reported to contain dioscin, protodioscin, methylprotodioscin, indioside D, and solamargine. Incanumine, a steroidal alkaloid glycoside from S. incanum, has exhibited strong cytotoxic property against human PLC/PRF/5 cells in vitro [11].
Since the isolated phytoconstituents and extracts of different species of Solanum were found to be very active against several microorganisms and cancer cell lines, it motivated the authors to investigate the antimicrobial and cytotoxic potential of aerial parts of six different species of genus Solanum grown in Saudi Arabia along with estimation of rutin ( Figure 1) by validated UPLC-PDA method.

Chemicals and Reagents.
The HPLC grade methanol and acetonitrile were purchased from Fisher Scientific, UK. Double-distilled, molecular grade water was obtained using a Millipore Milli-Q5 (Bedford, MA, USA) water purifier. All the solvents (HPLC grade) and solutions were filtered through membrane filter (Millipore-Millex-HV5 filter units, Durapore-PVDF5, polyethylene, 0.45 m pore size) and ultrasonicated for degassing before use. Other reagents used in the present study were of AR grade.

Instrumentation and Chromatographic Conditions.
The ultra-performance liquid chromatography (UPLC) was performed on the gradient system of Agilent Technologies (1290 Infinity) equipped with G4220A Infinity Binary Pump and Autosampler, G1316C Thermostat Column Compartment and Fraction Collector with G4212A PDA (Photodiode Array) Detector. The programming of the above UPLC configuration was done by ChemStation software. The separation of analytes was carried out on Agilent ZORBAX Eclipse XDB 80Å C 18 column (4.6 × 100 mm, 3.5 m; Agilent, California, USA) through gradient method by using acetonitrile and water as mobile phase. Other instruments used included vortex (VELP Scientifica, Italy), Mikro 200R centrifuge (Hettich Lab Technology North America), and Power Sonic 405 sonicator (Korea). All the analysis was performed at 25 ± 1 ∘ C temperature. The flow rate and injection volume were set at 0.18 mL/min and 10 L, respectively. The solutions and the mobile phases were sonicated for 30 min before use and UVdetection of rutin (analyte) was done at 332 nm.

Extraction of Plant Material by Ultrasonic-Assisted
Method. The aerial parts of S. schimperianum (SS), S. villosum (SV), S. coagulans (SC), S. glabratum (SG), S. incanum (SI), and S. nigrum (SN) were air dried, powdered, and passed through a 0.75 mm sieve. The extraction process was accomplished in a Transsonic-460/H ultrasonic cleaner (ELMA, Germany). The powdered plant materials (20.0 g, each) were extracted for 30 min by ultrasonication (20 kHz, 240 W), using ethanol (95%) [12]. The obtained ethanol extracts of the six Solanum species (SSEE, SVEE, SCEE, SGEE, SIEE, and SNEE) were centrifuged at 5000 rpm for 20 min and filtered. All extracts were concentrated and dried under reduced pressure using rotary evaporator (R-210, BUCHI). The estimated yields (w/w) of SSEE, SVEE, SCEE, SGEE, SIEE, and SNEE were 9.71, 6.43, 5.92, 5.81, 3.75, and 8.52%, respectively. Protein preparation wizard of GLIDE was used for the assessment and refinement of protein structure before performing molecular docking. The structure of protein was prepared by removing water molecules, adding missing hydrogen atoms, assigning bond orders, creating zero bond order to disulfide bonds, and deleting any other heteroatoms expect respective ligands. Missing loops and any side chains were added using PRIME module of Schrödinger software. The protein was then optimized to create H-bond network and finally energy was minimized using the optimized potentials for liquid simulations 2005 (OPLS 2005) force field by setting a default constraint of 0.30Å root mean square deviation (RMSD).

Ligand Preparation.
The structure of rutin was drawn using 2D sketcher of Schrödinger Suite and optimized for docking by assigning the bond orders and angles using LigPrep module. In LigPrep module, the 2D structure of rutin was converted into 3D structure and the energy was minimized using OPLS2005. The ionization state of rutin was generated at pH 7.0 ± 2.0 with the help of Epik module of LigPrep, keeping other parameters to default values.  [19,20] using the following relation:

Grid Generation and Molecular
where ΔG is the binding energy, T is temperature, R is Boltzmann gas constant (R=1.987 cal/mol/K), and Kd is the binding affinity.

UPLC-PDA Analysis of Rutin in the Ethanol Extracts of Different Solanum Species
2.9.1. Standard Solutions. The standard rutin (>94% pure) was procured from Sigma-Aldrich (USA). The accurately weighed quantity of rutin was dissolved in 5 mL of methanol in a 50 mL capacity volumetric flask and made up to 50 mL with methanol to get the stock (50 g/mL, final). Further dilution of the stock solution was done to get the final concentration of 1 g/mL.

Sample Solutions.
About 400 mg of the extract (SSEE, SVEE, SCEE, SGEE, SIEE, and SNEE) was dispersed in 10 mL of methanol and sonicated for its complete and quick dissolution. Subsequently, in a 10 mL capacity volumetric flask exactly 1250 L of the obtained solution was transferred and the volume was made up to 10 mL with the binary mobile phase to get a final known concentration of 5 mg/mL.

Method Validation.
The developed analytical method for the quantification of rutin in the SSEE, SVEE, SCEE, SGEE, SIEE, and SNEE was validated as per ICH guideline 2005 [21]. The validation parameters such as specificity, linearity, sensitivity, accuracy, precision, and recovery were checked. System suitability was tested in the beginning of quantitative analysis.
(1) Specificity. The ability of an analytical method to differentiate among the substances to be analyzed and other components present in the samples is known as specificity of the method. Specificity of the developed UPLC method is confirmed by elution and separation of the analytes of interest even in the presence of other potential constituents and the matrix. The resolution among the intensity of peaks of the desired constituents present in the SSEE, SVEE, SCEE, SGEE, SIEE, and SNEE was determined by analyzing the chromatograms obtained for reference standard and sample solutions, and the resolution was computed and estimated through the ChemStation software.
(2) Linearity. The linearity of the method was accessed by using the obtained calibration curves (n = 3) with the standard solutions at seven varying concentrations ranging from 1 to 80 g/mL for rutin. The values of the peak area obtained against the concentrations of the analytes were subjected to the linear regression analysis by using Excel (Microsoft, 2010).
(3) Limit of Detection (LOD) and Limit of Quantification (LOQ). The sensitivity of the developed method was established in terms of detection and quantification of the analytes, which was done from the calibration curves of the rutin. The LOD and LOQ were calculated by using the following equations: where SD was considered as the standard deviation of the responses and S was the slope of the calibration curve. LOD of any analytical method is the detection of the lowest amount of an analyte in any sample that can be noticed and identified but not essentially quantified, while LOQ of an analytical method is the quantification of the lowest amount of an analyte in any sample that can be calculated accurately with appropriate precision.
(4) Interday and Intraday Precision. The precision of the developed UPLC method was evaluated by assaying the samples. The assay of the samples was carried out in triplicate (n = 3) by adding known amounts of the standard solutions to the samples, at three different concentration levels (10, 20, and 40 g/mL for rutin) of the initial concentration of the sample. The intraday and interday precisions were articulated as the observed concentrations with respect to the actual concentrations in terms of relative standard deviations (RSD).
(5) Recovery as Accuracy Study. Recovery of the analyte by using the developed UPLC method was retrieved by analyzing the obtained peak areas of six determinations at four different concentration levels (20,30,40, and 50 g/mL for rutin). The variations in the recovered amount were stated in terms of percentage (%) of the obtained concentrations of the standards as well as in terms of relative standard deviations (%RSD).  filtered through Milli-Q5 filter unit before injecting in the UPLC system.

Statistical Analysis.
The statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's test for the estimation of total variation in a set of data. Results were communicated in terms of mean values with ± SD where the probability (p < 0.01) of getting the result was considered as significant.     [25]. These differences could also be due to the differences in the chemical composition of these extracts as the secondary metabolites of plants have many effects including antibacterial and anticandidal properties. All Solanum extracts were further assayed for their biofilm inhibitory property against the P. aeruginosa PAO1 strain that demonstrated statistically significant reduction in biofilm as compared to the untreated control ( Figure 2). Similar reduction in biofilm of PAO1 has previously been described with the extracts of edible plants and fruits of Capparis spinosa and T. foenumgraceum [26][27][28].

Molecular Docking of Rutin with DNA.
In the present study, we have used in silico methods to probe the binding site of rutin on dodecameric DNA molecule and the molecular interactions involved in stabilizing the complex (Figure 3). The most preferred binding site of rutin on DNA was found to be located at the major groove of DNA with an overall binding score of -233.85. The complex was stabilized by one conventional hydrogen bonds OP2-atom with B: DA17 (2.30 A), two carbon hydrogen bonds between A: DT7: O4 and H19 (2.36Å) and H20 (2.35Å) of rutin molecule (Table 4). Moreover, the DNA-rutin complex was stabilized by one electrostatic interaction between rutin and B: DG16: OP1    role in cell viability, the Topoisomerase II is an important target for several anticancer drugs [30]. The docking complex of rutin with Topoisomerase II and the corresponding interacting amino acid residues are shown in Figure 4. It is evident that rutin formed a complex with Topoisomerase II and the interactions were stabilized by two hydrogen bonds and six hydrophobic interactions. Rutin formed hydrogen bonds with Ser148 and Lys157, while residues such as Ile125, Pro126, Val137, Leu140, Ile141, and Ala167 formed hydrophobic interactions. Other residues involved in the interaction were Asn91, Asp94, Arg98, Lys123, Hie130, Thr147, Ser148, Ser149, Asn150, Gly161, Gly164, Gly166, and Lys168. It is interesting to note that rutin also interacted with Mg 2+ which plays indispensable role in the catalytic activity of Topoisomerase II . The docking score or binding energy of rutin-Topoisomerase II interaction was estimated to be -6.901 kcal/mol in standard precision (SP) mode and -10.532 kcal/mol in extra precision (XP) mode which corresponded to the binding affinity of 1.15 × 10 5 /mol and 5.03 × 10 7 /mol, respectively (   the extracts strongly supported the promising anticancer potential of Solanum species.

Molecular Docking of Rutin with Bacterial DNA Gyrase
B. DNA gyrase and its homologs are present in prokaryotes and some unicellular eukaryotes, thus making it a good target for antibiotics. DNA gyrase has two subunits (A and B) that together bind to DNA, hydrolyze ATP, and introduce negative supertwists during replication. Two classes of natural anti-gyrase B aminocoumarins and quinolones are already reported [31] that work by competitive inhibition of energy transduction of DNA gyrase through ATPase activity. Docking of rutin with bacterial DNA gyrase B resulted in the generation of multiple poses of low energies, of which the best poses with lowest energies were further analyzed for the protein-ligand interactions. We compared the root mean square deviation (RMSD) between the published crystal pose and docked pose that was within the acceptable limit of 2.0 A ( Table 6). The docked complex of rutin with gyrase B and the corresponding interacting amino acid residues are shown in Figure 5. It was evident that rutin formed a complex with gyrase B and the interactions were stabilized by seven hydrogen bonds and seven hydrophobic interactions. Other residues involved in the interaction were Gly75, Gly77, Lys110, Gly119, Arg136, and Thr165. Rutin formed two hydrogen bonds each with Asp46 and Asp73, and one hydrogen bond each with Asp49, Glu50, and Arg76. The docking score or binding energy of gyrase B-rutin interaction was estimated Evidence-Based Complementary and Alternative Medicine 9  in SP (ΔG = -7.065 kcal/mol) and XP (ΔG = -9.191 kcal/mol) modes which corresponded to the binding affinity of 2.13 × 10 6 /mol and 5.51 × 10 6 /mol, respectively. Moreover, we also estimated the MM/PBSA energies to accurately determine the strength of ligand-protein interaction (ΔG = -88.521 kcal/mol). In line with the in silico data on strong rutin-DNA gyrase B interaction, the high content of rutin in the extracts endorsed the marked antimicrobial activity of Solanum species.

UPLC-PDA Analysis of Rutin in Alcoholic Extracts of Different Solanum Species Optimization of Chromatographic
Condition. Varying ratios of acetonitrile (Solvent-A) and water (Solvent-B) were used as mobile phase, column oven temperature, detection wavelength, and mobile phase flow rate were investigated for better separation of the analyte. A gradient program was employed for the separations of the active biomarker in one run within the reasonably short run time. The gradient was set up to achieve a linear increase of  solvent-A from 0% to 10% for first 2 min followed by a steeper gradient, 10 to 35% of solvent-A in the next 3 min. Again the gradient was decreased from 35% to 20% in the next 2 minutes and finally, an additional 2 min of separation was extended with 100% solvent-B. The UV-detection wavelength of 332 nm was optimized and set as per the absorption maxima ( max ) for rutin. The developed method was found to furnish compact, sharp, and intense peak of rutin at the retention time (R t ) = 4.172 min ( Figure 6) with high resolution baseline.
3.6.1. Method Validation. Calibration curves were linear over a large concentration range of 1-80 g/mL for rutin. Calibration curve exhibited good linear regression with coefficient of correlation (r 2 ) value 0.9964 for rutin and recorded in Table 7. The LOD and LOQ obtained (Table 7) through the developed UPLC method were found as 0.015 and 0.047 g/mL, respectively, for rutin. The numerical values obtained for the intraday and interday precision were documented in Table 8. The developed method was found to be precise as the relative standard deviation (RSD) values for repeatability of intraday and interday precision studies were below 2.0%, which is under the limit as per recommendations of the International Conference on Harmonization guidelines. The recoveries in percentage and %RSD were deliberated from the numerical values of the y-intercept and slope of the obtained calibration curve. The calculated recovery values (Table 9) were found in the range of 98.67-99.20% for rutin, confirming the accuracy of the developed method. When the wavelength was intentionally changed from 330 to 334 nm, no any obvious effect was detected in the chromatogram, and no significant difference was found in the peak area and in retention time (Rt). However, there was a littlevariation in retention time (Rt) obtained when the flow rate was varied from 0.16 to 0.20 mL/min. The low calculated values of standard deviations and %RSD with practically unaffected Rt values for rutin subsequently with small careful and deliberate changes as mentioned above indicated the robustness of the developed UPLC-PDA method (  (Table 11). It was clearly evident from the result that, out of these six species, S. coagulans (Figure 7(a)) contained highest amount of rutin followed by S. glabratum (Figure 7(b)), S. incanum (Figure 7(c)), S. schimperianum (Figure 7(d)), S. nigrum (Figure 7(e)), and S. villosum (Figure 7(f)).
This might be considered as a maiden report on comparative analysis of rutin content in different Solanum species by using a precise, economical, and perfect UPLC-PDA method. Rutin has a high therapeutic value because it relieved the symptoms of several diseases like lymphatic and venous insufficiency, hemorrhagic diseases, and hypertension and due to its excellent hepatoprotective effect [32]. Though the cytotoxicity of rutin on cultured human cells is still debated, its marked cytotoxicity on different cancer cell lines has been widely reported [33][34][35]. It was found to induce apoptosis in prostate cancer cells, LNCaP at low concentration and in liver cancer cells, and HepG2 at very high concentration by acting as prooxidant instead of an antioxidant. It also inhibited the tumor cell growth by blocking cells amelioration through G0-G1 transition in HTC cells (rat hepatoma) by reducing the cells survival [36]. It is also reported to possess an excellent antibacterial property against E. coli by inhibiting the action of Topoisomerase II as well as its selective promotion of E. coli topoisomerase IV-dependent DNA cleavage, essential for viability [37]. In view of this, we have evaluated the anticancer and antibacterial properties of rutin only as it is one of the major components of Solanaceae family. In this report, high rutin content of S. coagulans supported its strong cytotoxic property against HepG2 and HEK-293. The moderate cytotoxic property of S. incanum and S. schimperianum may be attributed to its rutin content but the strong cytotoxic property of S. nigrum (SNEE) might be due to the synergistic effect of rutin along with other cytotoxic phytoconstituents like solamargine and protodioscin present in S. nigrum. However, elucidating the synergistic effect of other alkaloids with rutin is beyond the scope of this study. The highest zone of inhibition and low MIC value of SCEE, containing the highest amount of rutin, indicated the antimicrobial potential of rutin. In addition, our in silico

Conclusion
In the present study, our results justified the screening of different species of genus Solanum traditionally used in folk medicine to treat several ailments like microbial infections and cancer. The aerial parts of S. coagulans and S. nigrum possessed significant cytotoxicity against breast and liver cancer cells compared to S. schimperianum and S. glabratum. On the other hand, S. coagulans extract was highly cytotoxic against kidney cancer cells compared to S. schimperianum. The extracts of all the Solanum species were found to possess marked antimicrobial activities against the tested strains. Further, the estimated high content of rutin in the S. coagulans supported its potent cytotoxic as well as antimicrobial properties. Since Solanum species contains numerous phytoconstituents which were proved to be highly cytotoxic and antimicrobial agents, our data warrants isolation of cytotoxic and antimicrobial compounds. Also, the developed UPLC-PDA method used to quantify rutin in Solanum species can be employed in the profiling of other genera and quality control of marketed herbal formulations.

Data Availability
All the required data are available in the manuscript.

Conflicts of Interest
The authors declare that they do not have any conflicts of interest.