Comparative Insights into the Antimicrobial, Antioxidant, and Nutritional Potential of the Solanum Nigrum Complex

: Solanum nigrum is a traditional medicinal plant renowned as a cure for many diseases due to the presence of bioactive compounds. The Solanum nigrum complex refers to a group of more than 30 closely related but morphologically distinct taxa. Five indigenous taxa of this complex were investigated for their medicinal potential by using methanolic extracts. The efficacy of each plant was different for each of the seven bacteria studied. On comparing the MIC values, S. americanum was found to be most potent against Bacillus licheniformis (34 µg/mL), S


Introduction
Nightshades are a variety of plant species that belong to the genus Solanum, including many morphological variants collectively known as the Solanum nigrum complex [1,2].Being part of the family Solanaceae, all plants of the S. nigrum complex are famous for their historical use in herbal medicine.Traditionally, they have been applied for the treatment of many ailments, including dysentery, joint and muscle pains, ulcers, infections, fevers, and eye infections, and are reported to have laxative, diuretic, sedative, emollient, and tonic effects [3][4][5][6].These medicinal properties are attributed to the presence of significant concentrations of secondary metabolites, including alkaloids, flavonoids, carotenoids, saponins, and triterpenes [7][8][9][10].
S. villosum Mill., S. retroflexum Dunal, S. nigrum L., S. chenopodioides Lam., and S. americanum Mill.were the five indigenous taxa of the S. nigrum complex selected for study.The pharmacologically active compounds reported in the investigated plants included α-solanine, α-solamargine, β-solamargine, solasonine, solanidine, solasodine, squalene, phytol, and glycosides of quercetin [10][11][12][13].S. nigrum is a commonly occurring weed, but is also used as a food in the form of cooked vegetables or soup.The presence of polyphenols, polysaccharides, glycoproteins, and glycoalkaloids makes the plant active against various ailments, although glycoalkaloids can be toxic when taken in large amounts [14].
Studies have shown that black nightshades have in vitro anticholinesterase, antimonoamine-oxidase, and antioxidant characteristics that enhance their importance against neurodegenerative diseases [14].The fruit juice of ripe berries of S. nigrum has been reported to be active against both Gram-positive and Gram-negative bacteria, due to the presence of active phytochemicals [15].Reports in the literature have been given on the antibacterial and antioxidant activities of S. nigrum L, but the medicinal value of individual morphological and chemical variants has not been explored.The authors have previously worked on the chemotaxonomy of the Solanum nigrum complex [12,13].The present study includes the intensive in vitro study of five indigenous plants of the S. nigrum complex, focusing on the antibacterial, antioxidant, and nutritional potential of the plant.

Experimental Section
Five plant samples were collected from the Botanical Garden of GC University Lahore, Pakistan, and were identified by a taxonomist (Supplementary Materials, Table S1).The dried powder of leaves of the five taxa was extracted separately using methanol.The desolvated sample extracts were used to prepare 3 concentrations (1000, 2000, and 3000 µg/mL) in distilled water.Seven microorganisms (Bacillus subtilis PCSIR-B-248, Bacillus licheniformis PCSIR-B-252, Escherichia coli PCSIR-B-67, Salmonella typhimurium ATCC-14028, Nocardia asteroides PCSIR-B-178, Micrococcus luteus NRRL-B-287, and Proteus mirabilis ATCC-29245) were used to test the antibacterial activity of three concentrations of these five extracts via the method of Negi et al. [16].All of the bacterial strains were cultured at 37 °C in nutrient agar by taking the preculture from stock slants stored in the refrigerator at 4 °C.The bacterial culture (1 mL) was added to nutrient broth (9 mL) and incubated at 37 °C in a shaker to prepare the inoculum.Nutrient agar (20 mL) was allowed to settle in Petri dishes, and inoculum (0.5 mL) was added.The samples, along with three standard positive controls (benzyl penicillin, ampicillin, and streptomycin) and pure solvent as a negative control, were added to Petri dishes and incubated at 37 °C for 24 h.Readings for the zone of inhibition (mm) were taken in triplicate, and the minimum inhibitory concentrations (MICs) of the plant extracts were calculated by regression analysis.The antioxidant potential of the extracts was evaluated by applying reported protocols of the total phenolic content assay [17], lipid peroxidation assay [18], FRAP assay [19], ABTS assay [20], DPPH radical scavenging activity [21], and ferrozine metal-chelation assay [22].Proximate analysis of the plant material-including moisture, ash, fat, nitrogen and crude protein [23], crude fiber [24], carbohydrate [25], nitrogen-free extract [26], and trace elements [27] in the five taxa-was carried out according to the standard procedures.

Antibacterial Activity
The antibacterial activity of the five morphologically different variants of the S. nigrum complex was evaluated against three Gram-negative and four Gram-positive bacteria (Table S2).Among these five taxa, S. americanum was found to be the most active at all concentrations, since it provided the greatest inhibition against three bacteria: B. licheniformis (a human pathogen causing infections and food poisoning, especially in immunocompromised patients [28]), E. coli (the cause most of the infections in the digestive and urinary tracts of humans and animals [29]), and M. luteus (reported as a cause of meningitis in humans [30]).This suggests that S. americanum can be used for the treatment of several human diseases and infections.All taxa, except S. americanum, were moderately active against P. mirabilis, which is usually responsible for pneumonia, septicemia, urinary tract infections, and wound infections in humans [31].S. nigrum and S. retroflexum can be effectively used to treat typhoid fever, since they demonstrated significant activity against S. typhimurium [32].S. villosum was most potent against B. subtilis, which is a food contaminant [33], and N. asteroides, which results in pulmonary and cutaneous nocardiosis [34].The variation in the results shows that each taxon has its own bioactive contents, and should be used for specific infections.
The presence of alkaloids and flavonoids in Solanum plants plays a significant role in their use as herbal medicines [35,36].The minimum inhibitory concentrations (MICs) of the plant extracts (Table 1) showed that S. nigrum and S. retroflexum had the lowest MIC values against B. subtilis, M. luteus, and E. coli.It was observed that the MIC of S. retroflexum against E. coli (30 µg/mL) was even lower than that of benzyl penicillin (32 µg/mL).Another significant result was the MIC of S. villosum against P. mirabilis (44 µg/mL), which was close to that of the standard benzyl penicillin (33 µg/mL).

Antioxidant Activity
S. nigrum L. has been cited as having effective antioxidant properties [37][38][39]; however, the literature is silent on the antioxidant activities of the remaining four taxa under study.Hence, comprehensive research was conducted to determine and compare the antioxidant potential of these five taxa of the S. nigrum complex via six assays (Figure 1).Total phenolic content (TPC) is an indicator of the antioxidant potency of plants [40] and is the reason for peroxidation inhibition against the linoleic acid peroxidation system [41].A slight variation was observed in the TPC values (in the range of 20.30-26.47mg GAE/100 mg of dry weight) of the five taxa.The TPC of both S. americanum and S. nigrum was the highest amongst the taxa.Considerable antioxidant activity in the peroxidation system of linoleic acid was shown by all five taxa and was comparable to that of Trolox as the positive control.FRAP values depend on the presence of antioxidant secondary metabolites that vary in different plant species [42].The investigated samples had FRAP values of 5.78 to 8.45 mM FeSO4•7H2O/mL of extract, with the observation that S. americanum, S. nigrum, and S. villosum were relatively more active.The Trolox equivalent antioxidant capacity (TEAC) of each of the five taxa was determined via ABTS assay.S. retroflexum showed the highest TEAC value (33.88 mM/100 g DW), whereas S. americanum had the lowest (25.69 mM/100 g DW).Significant free radical scavenging activities exhibited by plants in the ABTS assay can be used as a systematic and accurate tool for assessing their total antioxidant potential on large scale [39].All five sample extracts showed moderate and similar DPPH radical scavenging activity.S. villosum had a slightly slow response, as observed from the kinetic curves.Moreover, the best EC50 (48.7 µg) and TEC50 (7 min) results were shown by S. chenopodioides (Table S3).
Metal-chelating activity is an assay reported for the determination of the concentrations of transition metals.The metal-chelating activity was observed for the plant extracts, and the results showed that they possess a chelating ability for capturing the ferrous ions before they can form a complex with ferrozine.The chelating potential of S. chenopodioides was greater than that of the other analyzed extracts, whereas that of S. americanum (42.361%) was the lowest.

Proximate Analysis
Proximate analysis and elemental composition help in the evaluation of the nutritional potential of medicinal plants [43].Xylopia aethiopica is recommended as a tonic in Ivory Coast due to its high contents of carbohydrates, fat, and crude fiber [44].The plant samples had high moisture content and produced small amounts of ash.Dietary fibers are composed of lignin, hemicellulose, and cellulose, which have certain physiological benefits for human health, and help in the absorption of trace elements in the gut while reducing the absorption of cholesterol [45,46].Our investigations revealed that the taxa of the S. nigrum complex are rich in dietary fibers (Table 2).Plant proteins not only provide amino acids for the synthesis of human proteins but can also provide energy [47].The reasonably good protein contents present in the analyzed samples supported their nutritional potential.Amino acids and proteins from S. nigrum have been investigated; however, the study did not specify the taxon used [47].The investigated plant samples can serve as part of a low-calorie, high-protein diet because of their low carbohydrate and fat contents.Kareav et al. [48] evaluated the nutritional value of one taxon of this group.Generally, the investigated taxa were found to be of good nutritional quality, and to be able to provide health benefits when used in appropriate proportions with the correct classification.Comparatively, S. nigrum can be recommended in food due to its relative high protein and dietary fiber contents.
There are certain minerals and trace minerals (required at less than 100 mg/day) in our body that are as essential as oxygen for our existence.They exist in our food as organic and inorganic compounds.Moreover, plants also take up heavy metals from the soil.The ashes of five taxa of the S. nigrum complex were analyzed for 10 minerals and trace minerals (Table 3).Calcium, magnesium, and potassium were detected in significant quantities, whereas toxic metals such as cadmium, chromium, and lead were detected in the least amounts.Minerals are often found as key components of bones, teeth, muscles, blood, nerve cells, and tissues.The role of each mineral is significant in performing various fundamental physiological processes, including digestion, message transmission in the nervous system, and cell metabolism.An imbalance of certain minerals can disrupt the assimilation of vitamins.For instance, zinc is required for vitamin A, calcium for vitamin C, and magnesium for vitamins of the B complex [49].Our results suggest that the investigated taxa have good nutritional value.

Conclusions
The five examined taxa showed different antibacterial activities against the seven tested bacteria.In some cases, the activities of the plant extracts were found to be comparable in effectiveness to commonly used antibiotics taken as standards.The MIC value of S. retroflexum was found to be lower than that of benzyl penicillin against E. coli.The five plant samples also showed significant antioxidant activities.The activity of S. nigrum was highest when the taxa were analyzed by TPC and FRAP assays, whereas S. chenopodioides showed the highest antioxidant potential in analysis by lipid peroxidation, DPPH, and metal-chelating assays.The overall antioxidant activities of S. nigrum and S. retroflexum were comparable.The presence of bioactive compounds resulted in significant antibacterial and antioxidant activities in the samples.These results suggest that the specific use of morphologically similar taxa can enhance the effectiveness of these traditional plants as cures for different diseases and infections.The functionality and chemical composition of individual plants can hence be determined by the systematic evaluation of their medicinal potential.These plant taxa can be used as healthy medicinal supplements when taken in the right proportions, due to their significant nutritional value.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/pr10081455/s1,Table S1: List of plants investigated with voucher numbers; Table S2: Zones of inhibition of different concentrations of methanolic extracts of S.

Table 1 .
MIC values of different taxa of the S. nigrum complex for the analyzed bacteria.

Table 2 .
Proximate analysis of the S. nigrum complex for its nutritional value.

Table 3 .
Determination of metals in the S. nigrum complex.