Evaluation of Antimicrobial and Chemopreventive Properties and Phytochemical Analysis of Solanum nigrum L. Aerial Parts and Root Extracts

: This study evaluated in vitro antibacterial, antifungal, anticancer and antioxidant activities of methanolic leaf and root extracts from Solanum nigrum L. and determined its chemical composition. The chemical proﬁle of S. nigrum L. extract was tested using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Disc diffusion and microdilution assays were used for the antibacterial activities. Antifungal activity was measured using the poisoned food technique. In vitro activity on the cell culture model was assessed by MTT assay, viability measurement and by determination of cellular ROS by DCFDA assay. The minimal inhibitory concentrations for extracts from aerial parts ranged from 125 to 500 µ g/mL for gram-positive cocci and Pectobacterium strains. The growth inhibition coefﬁcient ranged from 17–56%, depending on the dosage. The antifungal effect of S. nigrum extracts on the tested ﬁlamentous fungi depended on the dose. An inhibitory effect of 50–56% on fungi was observed against Alternaria alternata and Chaetomium globosum . The study showed a reduction in cellular vitality of THP1, A549, MCF7 and HeLa cancer lines using both extracts. In addition, there was a decrease in the number of viable cells in cultures incubated with the extract of aerial parts and a reduction in oxygen radicals in the cells. The obtained results indicate the possibility of using S. nigrum extracts from the aerial part as antimicrobial factors. Both extracts show chemopreventive properties by inhibiting the growth of cancer cells and reducing the level of free radicals. Both extracts show chemopreventive properties by inhibiting cancer cell growth and lowering free radical levels. The broad biological activity of the studied extracts can be used in agriculture, veterinary usages and medicine. (cid:48) (cid:48) and then oxidized in the presence of ROS (reactive oxygen species) to ﬂuorescent DCF (2 (cid:48) 7 (cid:48) -dichloroﬂuorescein). DCF was measured using a Varioscan LUX microplate reader at λ ex = 485 nm and λ em = 535 nm. Results were presented as E/E 0 , where E is the value of the test sample and E0 is the control value


Introduction
The search for new and effective antibacterial and antifungal agents is one of the key challenges of modern medicine and agriculture due to the ever-growing phenomenon of

Microbial Assay
The preliminary antimicrobial values from testing extracts were achieved using agar disc diffusion methods for tested extracts of S. nigrum ( Table 3). The extracts (100 µg/disc) of S. nigrum showed varying degrees of inhibitory effects. The zone of inhibition produced by the reference antibiotics (tetracycline or amphotericin B) was between 22.0 and 42.0 mm, 2-4 fold larger than that produced by the extract (0.0-11.5.0 mm). The maximum zone of inhibition was observed against plant pathogens of Pectobacterium strains (zone inhibitioñ [7][8][9][10][11].0 mm). The extract from aerial parts showed better antibacterial activity than from roots. The inhibitory effect was observed against nine bacteria, and against five in the case of the root extracts. Both extracts showed no inhibitory activity against B. cereus, P. aeruginosa, S. marcescens and Candida spp. An example of the zone of inhibition in the presence of aerial parts or root extracts of S. nigrum is shown in Figure 1. Table 4 showed the antibacterial activity of methanolic extracts from S. nigrum aerial parts and roots tested by serial dilution method determining MIC/MCB. Microbes used in this study are associated with various forms of infections in people, animals and plants. In our research, the extract of black nightshade showed antibacterial effectiveness at variable degrees, with MIC 90 values varying from 125 to <500 µg/mL. The extracts from aerial parts showed better antimicrobial activity against different bacterial strains than roots. Generally, extracts from aerial parts exhibit MIC ranging from 125 to 250 µg/mL, while extracts from roots have an MIC of usually 500 µg/mL or above. The lowest minimum inhibitory concentration was found against a plant pathogen from genus P. carotovora strain 1815 and 1822 (MIC = 125 µg/mL) and gram-positive cocci (MIC = 250 µg/mL). However, the bactericidal effect was observed at a 2-fold concentration (MBC = 250 µg/mL or MBC = 500 µg/mL, respectively). The extracts from the aerial parts also inhibited the growth of endospore-forming bacilli and E. coli but at a concentration of MIC = 500 µg/mL. The extract from roots did not exhibit activity against S. aureus, Bacillus spp., E. coli, P. aeruginosa and C. albicans at the tested concentrations.
C. parapsilosis KW-Cp1 0.0 ± 0.0 (0) 0.0 ± 0.0 (0) 33.3 ± 0.5 * reference control-tetracycline for bacteria and amphotericin B for Candida (30 μg/disc). ±SD (standard deviation). ( ) the percent inhibition of microorganisms compared to suitable reference control is in brackets (control = 100%).   Tables 5 and 6 show antifungal activity of the tested extracts of S. nigrum L. Most of the tested filamentous fungi did not react uniformly to the presence of black nightshade extracts. Aspergillus niger, A. versicolor, Epicocum nigrum and Fusarium oxysporum, depending on the dose and the extract, showed a slight acceleration or slowing down of the colony growth rate (Table 4). The species most stimulated by the extract from the aerial parts of black nightshade at a dose of 500 µg/mL was A. flavus. The coefficient of colonies' growth inhibition for this combination was −29 mm (growth stimulation). Root extract in the same dose did not strongly stimulate growth (H = −9 mm), and in lower doses, it inhibited its growth. Penicillium notatum was significantly inhibited by black nightshade root extract. Depending on the dosage, the growth inhibition coefficient ranged from 17-36% (Table 6). The same coefficient with the use of aerial parts extract did not exceed 9%. Bipolaris sorokiniana, after applying black nightshade root extract at a dose of 500 µg/mL, was inhibited at the level of 43%. The same dose of the extracts from the aerial parts inhibited the fungus by 20%. The highest antifungal activity of S. nigrum extracts was recorded in the case of Alternaria alternata and Chaetomium globosum. For both fungi, the growth inhibition coefficient value rose with the extract dosage increase, reaching 55 and 56, respectively, in the highest dose of 500 µg/mL.

Cell Culture
The effect on cell culture viability during the MTT assay in the biological activity assays is shown in Figure 2. In parallel, the evaluation of the number of viable cells (Figure 3) in the automated counter was also carried out to determine whether the effect observed in the MTT assay is related to cell growth inhibition or the cytotoxicity of the tested extracts. The tested extracts differ in their impact on cell cultures. In the case of normal cells and human fibroblasts (Figure 2a), an increase of 33% in the number of fibroblasts was observed when an extract from aerial parts was used at a concentration of 250 µg/mL. A greater increase in cell number is also seen at concentrations of 100 and 500 µg/mL. The increase in viability in the MTT test suggests a faster proliferation of fibroblasts and a possible pro-regenerative effect of the tested extract. No cell growth-promoting effect is seen for the other concentrations nor the root extract. The root extract has little inhibitory effect on cell vitality. For fibroblast cells, no impact on the number of viable cells can be seen in Figure 3.

Cell Culture
The effect on cell culture viability during the MTT assay in the biological activity assays is shown in Figure 2. In parallel, the evaluation of the number of viable cells ( Figure  3) in the automated counter was also carried out to determine whether the effect observed in the MTT assay is related to cell growth inhibition or the cytotoxicity of the tested extracts. The tested extracts differ in their impact on cell cultures. In the case of normal cells and human fibroblasts (Figure 2a), an increase of 33% in the number of fibroblasts was observed when an extract from aerial parts was used at a concentration of 250 μg/mL. A greater increase in cell number is also seen at concentrations of 100 and 500 μg/mL. The increase in viability in the MTT test suggests a faster proliferation of fibroblasts and a possible pro-regenerative effect of the tested extract. No cell growth-promoting effect is seen for the other concentrations nor the root extract. The root extract has little inhibitory effect on cell vitality. For fibroblast cells, no impact on the number of viable cells can be seen in Figure 3.   Results are presented as the ratio of the value obtained in the test to the control culture. Differences between results for tested plant extracts compared to control and between extracts (* p < 0.05). The other cell lines tested were cancer cell lines. The monocytic line THP1 is used as a model for the effect of compounds on cells involved in inflammation. We observed a decrease in cell viability in the MTT assay for this line. A greater effect was observed with the root extract. It already exerted an inhibitory influence on cells from the lowest concentration tested, and this effect was dependent on the concentration of the extract. In the case of the extract from aerial parts, the inhibitory effect was weaker and visible only at a concentration of 100 µg/mL. An impact on the number of living cells was found for the extract from above-ground parts (15% compared to the control).
For the other lung, breast and cervical cancer lines tested, the activity of both extracts was similar, and both showed an inhibitory effect on cancer cells. For the MCF7 line, the extract from the aerial parts shows less impact on vitality, especially at the highest concentration tested. The activity of both extracts is concentration-dependent. The reduction in vitality ranged, depending on the line, from 43% (A549) to 59% (MCF7) ( Figure 2). A decrease in the number of viable cells was also observed for the three cell lines ( Figure 3). This was clearly evident after incubation with extracts isolated from the aerial parts of the plant. In the case of the root extract, no such effect was observed. The results indicate that the observed effect in the MTT assay is due to a reduction in cell growth. Compared to the control, the decrease in cell viability is small, ranging from 9.4 (HeLa) to 11.9 (MCF7).
The effect of the tested extracts on the level of oxygen radicals ROS in fibroblast cultures was also evaluated during the cell model studies (Figure 4). First, tests were performed on cells incubated with the tested extracts. Then, tests were performed on cells not subjected to oxidative stress and incubated with 100 µM H 2 O 2 solution. In both cases, the effect of the tested extracts was observed, consisting of a reduction of ROS in the cells. The observed effect was concentration-dependent and occurred in cultures not subjected to oxidative stress and incubated with H 2 O 2 . In both variants, a greater impact was found for the root extracts. In resting incubation, a difference was observed for the 500 µg/mL concentration and for incubation with H 2 O 2 at 250 and 500 µg/mL concentrations. The decrease in ROS was 41% and 49% at maximum concentrations for the root extract, respectively. Appl

Discussion
This study determined the concentrations of compounds in the methanol extracts of S. nigrum in the above-ground parts and the roots. In the first study examined, the total flavonoid content determined by Aryal et al. (2019) [33] was 16.42 mg QE/g of dry extract, while in our study, we determined total flavonoid content to be 15.73 mg QE/g of dry extract. Regarding the root portion of S. nigrum, there are no reliable sources in the scientific literature reporting flavonoid content. Only for plants belonging to the same family, such as wolfberry (S. xanthocarpum) (Schrad. And Wendl), the total content of flavonoids in the root was 15.83 mg QE/g of the dry extract [34]. Moreover, our extracted compounds from S. nigrum, both from the ground and the root parts, partially coincide with the composition published by Wu et al. (2013) [35]. This study also identified naringenin, or tomatin compared to studies by Wu et al. (2013) [35]. All those compounds may be found in Solanum plants, including S. nigrum [35][36][37]. Different plant chemotypes can cause these differences, which are well documented in the scientific literature on plant

Discussion
This study determined the concentrations of compounds in the methanol extracts of S. nigrum in the above-ground parts and the roots. In the first study examined, the total flavonoid content determined by Aryal [35]. This study also identified naringenin,

Discussion
This study determined the concentrations of compounds in the methanol extracts of S. nigrum in the above-ground parts and the roots. In the first study examined, the total flavonoid content determined by Aryal et al. (2019) [33] was 16.42 mg QE/g of dry extract, while in our study, we determined total flavonoid content to be 15.73 mg QE/g of dry extract. Regarding the root portion of S. nigrum, there are no reliable sources in the scientific literature reporting flavonoid content. Only for plants belonging to the same family, such as wolfberry (S. xanthocarpum) (Schrad. And Wendl), the total content of flavonoids in the root was 15.83 mg QE/g of the dry extract [34]. Moreover, our extracted compounds from S. nigrum, both from the ground and the root parts, partially coincide with the composition published by Wu et al. (2013) [35]. This study also identified naringenin, naringenin-7-O-glucoside, kaempferol-3-O-rutinoside-7-O-glucoside, cyanidin-3-O-(6 )-O-α-rhamnopyranosyl)-β-glucopyranoside, del-finidin-3-O-(6 -O-α-rhamnopyranosyl)β-glucopyranoside, pelargonide-in-3-O-(6 -O-α-rhamnopyranosyl)-β-glucopyranoside or tomatin compared to studies by Wu et al. (2013) [35]. All those compounds may be found in Solanum plants, including S. nigrum [35][36][37]. Different plant chemotypes can cause these differences, which are well documented in the scientific literature on plant secondary metabolites [34]. Moreover, Wu et al. (2013) [35] do not state whether the roots were also used for extraction in their studies of plants, which may also be the reason for the noted differences.
Studies have shown the highest antimicrobial activity of compounds such as isoquercetin and naringenin and its derivatives: rutin, demysin, solamargine, solanidine, solanine, solasodine, solasin and tomatin, the activity of which has been repeatedly mentioned in numerous scientific reports [36][37][38][39][40][41][42]. All these compounds or groups to which they belong can be found in the concise review by Chauhan et al., (2012) [37] on the therapeutic role of S. nigrum, especially in terms of antiviral, antimicrobial, anti-inflammatory and antitumor activity. In addition, some plant protective actions, such as cardioprotective or hepatoprotective, have also been described. A similar but more comprehensive review report by Atanu et al. (2011) [36] also draws attention to the mentioned actions. Additionally, it points to the important role of solanine in terms of antitumor activity in S. nigrum extracts. Nevertheless, solanine and its related compounds, with a small amount of protein, are more often associated with toxic effects [39]. Despite its anti-cancer effects, the presence of solanine can cause headaches, colic, skin warming, nausea and some physical symptoms such as weakness or depression. Moreover, by focusing on the antimicrobial activity of  [44] indicated that some steroid compounds are responsible for the activity of other plants from the Solanaceae family; S. villosum can regulate the reproduction of S. aegypti, which may have a significant impact on the spread of dengue fever, dengue hemorrhagic fever and dengue shock syndrome.
Earlier reports highlighted that S. nigrum extracts exhibited biological activity against Gram-negative and Gram-positive bacteria as well as fungi (e.g., [37,[45][46][47][48][49]). The antimicrobial activity of S. nigrum extracts was tested in the concentration range from 25 µg/mL to 200 mg/mL and was found to depend on the qualitative and quantitative composition of the test extracts [7,37,[45][46][47][48][49]. In our research, the antimicrobial activity of the extracts of S. nigrum from aerial parts and roots was tested by the disc diffusion method and serial dilution method to determine MIC/MCB. Our results showed different degrees of inhibition of growth against the pathogens tested. The methanolic extracts showed a broad antibacterial spectrum against the tested bacteria with the diameter of the zone of inhibition varying from 4 to 11.5 mm (13-41% against the control). However, the minimum inhibitory concentration (MIC 90 ) ranged from 125 to 500 µg/mL. However, the extract from aerial parts showed a stronger antimicrobial effect than the extract from roots. Plant pathogens (Pectobacterium spp. and Erwinia spp.) and gram-positive cocci were the most sensitive to the tested extracts. The observed antibacterial activity is probably related to the contents of plants, including Solanaceae, including alkaloids, glycoalkaloids, tannins and terpenoids, which can penetrate the bacterial wall and/or cell membrane and have bacteriostatic or bactericidal effects by penetrating deep into the cell [45].
The tested extracts showed activity against cells of tumor lines by inhibiting cell growth. There are few reports in the literature on the activity of extracts obtained from S. nigrum. Ongoing research [30] showed activity with significant cytotoxicity in human endometrial cancer cells by activating autophagy and cell death. An aqueous leaf extract was used in the study; however, its composition is missing. The following paper on in vitro activity evaluates the antioxidant activity of methanolic and aqueous extracts. The extracts were partially characterized in terms of composition. SN leaf extracts could reduce oxidative stress in primary cultures of rat astroglial cells. In addition, the extracts showed antioxidant properties and neuroprotective effects. According to the authors, natural phenolic antioxidants in functional foods prevent neurodegenerative diseases. Another study investigated the extracts' antiproliferative activity against cells of leukemia lines. Furthermore, in this study, the activity of the extracts was confirmed [28].
In the study of antitumor activity, methanol and chloroform extracts from S. nigrum leaves and stems were also used [30]. The authors assessed the activity of the extracts in in vitro studies on the PC-3 human prostate cancer cell line and the HeLa cervical cancer cell line. Additionally, tests were carried out on normal cells. All extracts at a 100 µg/mL concentration showed anti-tumor activity and were not toxic to normal cells. This study confirmed S. nigrum in treating cancer in traditional medicine in Sudan [30].
S. nigrum is used in traditional medicine as an antiviral, anti-inflammatory and hepatoprotective agent [28,30,[50][51][52][53]. The cited literature data confirm the results obtained. The tested extracts show activity consisting in inhibiting cell growth. It is visible in MTT test results and approved by a direct cytotoxicity test (Figure 1). The results of the MTT test confirm the antiproliferative activity of the tested compounds, confirming the literature data. The compounds themselves show low cytotoxicity. The obtained results indicate new possibilities of anticancer application of extracts in the prevention of lung cancer or breast cancer using lines A549 and MCF7. Conducted research will be expanded in the future to evaluate the mechanism of extracts' examination on cells. Due to the high content of polyphenols, we planned to investigate the effects of extracts on the level of oxygen and nitrogen free radicals, and thus, on lipid peroxidation and potential DNA strand damage or on the possibility of intercalation with DNA. It is also planned to determine whether the tested extracts also affect pro-apoptotic proteins, e.g., p53 and whether there is a resulting increase in the number of cells in a state of apoptosis or only inhibition of cell division. Moreover, we are currently investigating the effect of adding these extracts simultaneously with doxorubicin, which is commonly used in therapeutic regimens for colorectal cancer. As is well known, the risk of multidrug resistance in colorectal cancer is high, so it is important to look for substances that can prevent the release of cytostatic from cancer cells. The results obtained in this study are promising and allow for a deeper understanding of the actions of the obtained mixture of compounds in extracts from the upper part of the plant and the roots and of their fractions separately to determine the potential synergistic effects. Pelargonidin is particularly noted when assessing the polyphenols' composition in the tested extracts. This compound is present in high amounts in both studied extracts, especially in the extract from the root. This compound shows strong anticancer effects through apoptosis and induction of cell cycle arrest. The high contents of this potent compound may explain the chemopreventive activity of the studied extracts [54,55].
Extracts from S. nigrum are used in traditional medicine as anti-inflammatory agents. This may be related to their effect on the proliferation of inflammatory cells, such as monocytes. Therefore, the THP1 line is a good model to study the anti-inflammatory activity of the compounds. The results obtained showed growth inhibition of this cell line as well.
The results obtained (Figure 4) also confirm the above antioxidant properties. The tested extracts can have a protective effect on cells by reducing the level of oxygen free radicals. Importantly, their impact was observed in cells not subjected to oxidative stress and those incubated with H 2 O 2 . Due to the higher polyphenols content, the extract from aerial parts shows better activity.
The extract from aboveground parts also stimulated fibroblast proliferation. The effect was not large, but it was nevertheless intensive. The enhancement of the growth of normal cells may explain the pro-regenerative impact of the extracts used in traditional medicine.

Plant Material and Extract Preparation
Plant material for the preparation of bioactive methanol fractions of the Solanum nigrum L. was collected in June 2019 from a maize field in Malin, Poland (51.220534, 17.059176). Complete shoots and roots of plants obtained from the field were cut into small pieces and then subjected to drying in a drying cabinet at 25-30 • C for 72 h. The relative humidity of the milled material was <10%. Dried aerial parts and roots were ground in a grinder (IKA POL M20 Werke) and extracted with absolute methanol (ratio: 1:5, w/v). Further, the mixture was shaken for 60 min on a rotary shaker (125 rpm). Finally, the crude extracts were filtered and dried with a rotary evaporator (VWR IKA). The dry extracts were weighed and stored at 4 • C until tested.

Total Flavonoid Content
Total flavonoid compounds concentration in S. nigrum aerial parts and root extracts were determined according to the method described by Aryal et al. (2019) [33]. Briefly, 1 mL of plant extract (20-200 µg/mL), aluminum chloride solution (10% w/v), 0.2 mL potassium acetate (1 M) and 5.6 mL distilled water were added together, and the mixture was prepared for 30 min. After that, the samples were subjected to absorbance read with Cintra 303 spectrometer (GBC Scientific Equipment Ltd., Dandenong, Australia) at 415 nm. The results were expressed as mg of quercetin equivalents (QE).

LC-MS/MS Analysis
Methanol extracts (5 mg/mL) from the aerial parts and roots of S. nigrum were diluted 1000 and 100 times with Chromasolv grade methanol (Sigma Aldrich, Steinheim, Germany). Then 100 µg of luteolin (Sigma-Aldrich, Steinheim, Germany) was added as an internal standard. The obtained dilutions were then filtered through Phenex-RC syringe filters (Phenomenex, Torrance, CA, USA) and subjected to chemical profiling according to the procedure developed by Wu et al. (2013) [35], which allowed for a comprehensive study of the chemistry of Solanum species. Compounds belonging to the flavonoid, steroid or glycosides, glycoalkaloids or alkaloids were selected for screening as they have the greatest potential for antimicrobial or antioxidant activity. The screening was performed on an LCMS-8045 instrument (Shimadzu, Kyoto, Japan) equipped with a Kinetex 2.6u C18 100A 100 × 3.0 mm column with a 3 mm ULTRA shell (Phenomenex, Torrance, CA, USA).
The mobile phase used for the chromatographic separation consisted of 0.1% aqueous formic acid (A) and methanol (B). A solvent gradient was applied from 10% to 80% B in 0-3 min, 80% B in 3-15 min and 80 to 10% B in 15-20 min. The flow rate and injection volumes were 0.3 mL/min and 10 µL, respectively. The column temperature was set to 40 • C.
Mass spectrometry was performed by electrospray ionization (ESI) under the following conditions: Q3 SIM (m/z details are given in Table 1 in positive mode); nitrogen for nebulization (flow 3 L/min) and desolvation (temperature 526 • C); both heating as well as a drying gas flow of 10 L/min; interface; DL and heating block temperatures of 300, 250 and 400 • C, respectively.
Flavonoid quantification was based on normalization of the peak area based on the peak area of the internal standard. The analyses were carried out in triplicate.

Agar Disc Diffusion Assay
The disc diffusion method measured the antibacterial activity of methanolic extracts [56]. First, 100 µL of bacterial suspension containing 1.5 × 10 8 CFU/mL or yeast suspension containing 1.5 × 10 6 CFU/mL was spread on Mueller Hinton agar (MHA, Sigma-Aldrich, St. Louis, MO, USA) or Sabouraud dextrose agar medium (SDA, BTL, Łódź, Poland), respectively. The turbidity of the tested strains was standardized to 0.5 McFarland (spectrophotometer VIS-723G, Rayleigh, Beijing). Then, paper discs (Whatman no. 1, England, 6 mm diameter) were placed on the agar surface and impregnated with 30 µL of stock solutions of 100 µg/mL. For a negative control and a solvent, 10% DMSO was used. In addition, filter discs impregnated with 30 µg/mL tetracycline (Sigma-Aldrich) or amphotericin B (Gibco, Life Technologies, USA) were used as a positive reference control for bacteria and fungi, respectively. The plates, after remaining at 4 • C for 2 h, were incubated at 37 • C (24 h) for bacterial strains and at 30 • C (48 h) for yeasts. Antimicrobial activity was evaluated by measuring the diameters of inhibition zones in millimeters. The experiment was done in triplicate, and mean values are presented ( Table 2).

Determination of Minimum Inhibitory Concentration, Minimum Bactericidal Concentration and Half Inhibitory Concentration
The minimum inhibitory concentrations (MICs) were determined by a serial dilution method [57]  where: ODpc is the mean value of the measured optical density of the positive control.
ODs is the mean value of the measured optical density of the extracts of the test sample. Minimum bactericidal concentration (MBC) was defined as the lowest extract concentration that killed 99% of microorganisms. To determine MBC, cultures were taken from each well without visible growth and inoculated on MHA.

Antifungal Activity
Antifungal activity was measured using the poisoned food technique [58]. First, methanolic extracts dissolved in 10%DMSO were added to the liquified PDA medium (cooled to 50 • C) to obtain 50, 100, 250 and 500 µg/mL concentrations. Then the medium was poured into Petri dishes. After solidifying the medium with the addition of extracts, injections were done with discs (8 mm diameter) cut out from the edge of an actively growing one-week-old colony and placed in the center of test Petri dishes. In the case of Aspergillus and Penicillium, 20 µL of spore suspensions were used as inoculum. The concentrations of the spore suspensions were determined in a hemacytometer and adjusted to 2.5 × 10 6 spores per mL. Linear growth of the mycelium was measured for nine days, every 24 h. Incubation was carried out at 24 • C. The control contained diluents (DMSO) at a dose of 50, 100, 250 and 500 µg/mL. All tests were done in triplicate, in two series.
The obtained results were expressed in the linear growth index and the colony growth inhibition or stimulation coefficients. The linear growth index is calculated using the following Formula (2) [59]: where: T-linear growth index, A-average measurements of colony diameter (mm), D-duration of the experiment (days), b1-bx-the diameter increase of the colony since the last measurement (mm), d1-dx-the number of days since the previous measurement.
Coefficient of inhibition (H) or stimulation (S) of the radial growth of fungi-H (%) calculated by the modified Abbott Formula (3) [59]: where: H-coefficient of radial growth inhibition of the fungus, K0-the diameter of the colonies on the control Petri dish, F-the diameter of the colonies on the Petri dish containing certain levels of plant extract.

Cell Lines and Conditions
Five human cell lines were used in this study, which provides a good model for in vitro assessment of cytotoxicity and anti-tumor activity. In addition, studies were also performed on a monocytic line often used to study inflammation.

Cell Culture Tests
Each cell line was cultured from thaw for 2 weeks before testing. The cells were detached with trypsin/EDTA solution to start the assay. Medium containing 10% FBS was used to neutralize the effect of the trypsin/EDTA solution. The cells were then centrifuged (200× g), stained with 0.4% trypan blue solution, counted, and their viability was assessed in a Juli Br cell counter. Finally, the cells were seeded into 24 or 96-well plates and incubated in a CO 2 incubator for 24 h at 37 • C. The number of cells was a sub-template of 1 × 10 4 or 2 × 10 3 cells per well. Test compounds at different concentrations (25-500 µg/mL) were added to the cells. The cultures were incubated for another 24 h. After this time, further tests were performed [60].

MTT Assay
The MTT assay was used to determine the effect of the tested extracts on the metabolic activity of the tested cell lines. The test results allow for the determination of the influence of tested compounds on the vitality or growth of cell cultures. Cells were incubated with the tested plant extracts for 24 h. After removing the medium with the test compounds, 1 mg/mL of MTT solution in MEM without phenol red and FBS was added to the plate (to each well), and the plates were incubated at 37 • C for 2 h. The medium was again removed, and the formazan crystals were dissolved in isopropanol. A MultiscanGo microplate reader (Thermo Scientific, Waltham, MA, USA) measured absorbance at 570 nm [61].

Cell viability
Cell viability was assessed after 24 h of incubation of the test extracts with cell cultures. After incubation in 24-well plates, cells were harvested into tubes as previously described using trypsin solution for detachment. The resulting cell suspension was centrifuged at 200× g for 5 min. The pellet was then resuspended in PBS. Samples were then transferred to chips and analyzed in a NucleoCounter ® NC-200 (ChemoMetec A/S, Allerod, Denmark). Viability and quantity were measured using Via1-Cassette™ cassettes for cell sampling and staining. Acridine orange (AO) and 4 ,6-diamidino-2-phenylindole (DAPI) fluorophores, immobilized in the cassette channels, were used for staining.

Measurement of Intracellular ROS Levels
Tested plant extracts were added to the cell cultures and incubated with the cells for 4 h. The cells were then washed and incubated with DCFH-DA (non-fluorescent probe) in the dark at 37 • C for 2 h in CO 2 . The non-fluorescent, non-polar probe DCFH-DA (2 7dichlorodihydrofluorescein diacetate) at a concentration of 25 µM was used as an oxidative stress marker to determine intracellular ROS levels. After this time, cells were washed twice with PBS, and PBS with 100 µL H 2 O 2 was added (to a portion of the plates) for 30 min. During incubation, DCFH-DA inside the cells was hydrolyzed by esterases to a polar, non-fluorescent DCFH (2 7 -dichlorodihydrofluorescein) and then oxidized in the presence of ROS (reactive oxygen species) to fluorescent DCF (2 7 -dichlorofluorescein). DCF was measured using a Varioscan LUX microplate reader at λex = 485 nm and λem = 535 nm. Results were presented as E/E 0 , where E is the value of the test sample and E0 is the control value [62,63].

Statistical Analysis
All results in the tables are presented as mean ± SD (standard deviation) relative to the control (E/E 0 ). E is the culture with the test compound, and E 0 is the negative control without the compound. Statistical methods used were two-way ANOVA analysis of variance and Tukey's post-hoc test. The point of significance was established * p < 0.05. Statistical analyses were performed with Statistica v.13 software.

Conclusions
The obtained results showed that the extracts of Solanum nigrum L. from its aerial parts possess antimicrobial properties that can be used as a natural antimicrobial agent for infectious diseases of humans, animals and plants. The extract from aerial parts had the most efficient bactericidal activity against plant pathogens of the genus Pectobacterium (MBC = 250 g/mL). The most efficient fungistatic effects of S. nigrum extracts were observed against the fungi A. alternata and Ch. globosum (50-56% inhibition). Both extracts show chemopreventive properties such as anticancer and antioxidant activities. The activity is due to the high content of isoquercetin, naringenin and its derivatives, rutin, demissine, solamargine, solanidine, solanine, solasodine, solasonine and tomatine; however, further analysis is needed to correlate the obtained results with the phytochemical composition of the organic extracts from the aerial parts of the nightshade.