Bactericidal, Antioxidant Activity and In Silico Analysis of Phytochemicals Derived From Selected Plants of Solanaceae Family

A diverse range of active therapeutic constituents i.e. antioxidants from medicinal plants of Solanaceae family are responsible for anticancer & antibacterial activity. The study was aimed to investigate phytochemicals i.e. alkaloids, phenols, tannins, flavonoids, saponins, glycosides, and steroids qualitatively in selected plants of Solanaceae family. Total phenolic and flavonoid content of the aqueous extracts of the plants were determined quantitatively by the Folin-Ciocalteus reagent method and modified Aluminium Chloride assay respectively.Flavanoids present in the extracts of Solanumnigrum (fruit and leaf), Daturainoxia (leaf) and Capsicum annum (fruit) samples were 0.39 ± 0.030, 0.42 ± 0.020, 0.51 ± 0.04, 0.34± 0.030µgmL -1 respectively, whereas phenolic contents were 94± 1.2, 107± 1.5, 96± 1.3, 87± 1.9 mg chlorogenic acid equivalent respectively. The aqueous and ethanolic extracts of S. nigrum , D. inoxia and C. annum showed bactericidal activity against strains of Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia and Staphylococcus aureus with (MIC)2mgmL -1 and 5mgmL -1 . Highest free radical scavenging of DPPH activity 81.3% was exhibited by 2mgmL -1 methanolic extract of D. inoxia. Docking of Murine with Saponin, Flavone, phenol showed hydrogen bond of length 2.47 Å, 1.98 Å, 2.285 Å respectively while two H-bonds with 1.96 and 2.56 Å bond lengths of alkaloids. Representative trends and in silico assay results explain the synergism among flavonoids, alkaloids, and phenols. The phytochemicals provide antibacterial activity, antioxidant property as well as anti-cancerous bioactive compounds such as alkaloids and tannins, justifying their therapeutic efficacy in the field of cancer medicine.


Introduction
The ethnobotanical research is generally based on phytochemical information, which is considered as an effective approach in the discovery of new pharmaceutical agents from plants.Phytochemicals present a variety of biological properties due to their significant radicals scavenging effect.Polyphenols including (phenolic acid, flavonoids, and tannins) showed antitumor effects as proposed by (Gogoi & Islam, 2012).Around the world, family Solanaceae is distributed in the hot as well as moderate zones.It comprises the large genus Solanum, which contains steroids like glycoalkaloids, very important bioactive substances ecologically, and to the commercial point of view.Steroidal glycoalkaloids are synthesized by the plants for protection against pests and pathogens, nominated as defensive substances (Nino, Correa, & Mosquera, 2006).Plant members of the Solanaceae family are rich in these bioactive chemical substances such as alkaloids, tannin, flavonoids, and phenolic compounds and have attained much attention by researchers as they impart pharmacological properties of anticancer, antioxidants and antitumor (Gogoi & Islam, 2012).
Ethno-pharmacological relevance: Globally nutritional use of bell pepper is well recognized, yet the health relating benefits and medicinal properties are enormous.Vitamins and minerals possess antimicrobial and anticancerous properties.Chilli pepper (Capsicum annum) ingredients are effectively used locally for cancer, rheumatoid arthritis, bronchitis, chest colds, cardiac arrhythmias, headache, and numerous sicknesses.Chilli contains capsaicin, an ingredient employed for pharmaceutical creams, gels, and plasters, the essential oil extracted from the pods of capsicum, according to (Saleh, Omer, & Teweldemedhin, 2018).Datura stramonium has been scientifically confirmed to contain tannins, alkaloids, polysaccharides, and proteins.D.
stramonium/anoxia plays a further role in stimulating the central nervous system, as a decongestant, skin infections, treatment of toothache and in the treatment of baldness.Profound antibacterial activity in leaves and seed extract of D. anoxia were recorded as published by (Mahmood, Mahmood, & Mahmood, 2012).Jain et al (2011) narrated S. nigrum(mako or black nightshade) is mainly used in the composition of many medicines to cure pain, fever, inflammation and as antibiotics (Jain, Sharma, Gupta, Sarethy, & Gabrani, 2011).This ethnobotanical research involved the quantitative determination of phytochemicals from the crude extracts of plants.The antioxidant compounds show high free radical scavenging activity which accompanied their bactericidal efficacy also.The bioinformatics study involved structural analysis of proteins and phytochemicals.

Aqueous Extract
The aqueous extract was prepared by taking 50g of plants part.Leaves or fruit were crushed and ground into 10 mL distilled water while stirring.After overnight incubation at room temperature, the mixture was stirred through magnetic stirrer for one hour and filtered for further qualitative tests (Bashir, Mohammed, Magsoud, & Shaoub, 2013).

Qualitative Phytochemical Screening:
2.5.1.Alkaloids: Mayer's Test was adopted for alkaloids presence (Balabhaskar & Vijayalakshmi, 2015).Mayer's Test: To 1 ml of the plant extract, concentrated hydrochloric acid 2 mL was added.Then few drops of Mayer's reagent were added into it.White or green color precipitates specified the presence of alkaloids.(Mayer's reagent: It is a mixture of two solutions.Mercuric chloride and potassium iodide aqueous solutions).
2. Potassium iodide (5 g) added into 200 mL of distilled water, (after mixing both solutions the final volume was made up to 100 ml with distilled water).

Saponins:
Crude extract prepared in distilled water was vigorously shaken in distilled water (5mL).The formation of stable form specifies the saponin's presence (Yadav and Agarwala, 2011).

Tannins:
Lead acetate Test: To 1 mL plant extract,5 drops of 1% lead acetate along with 10 mL distilled water were added.White precipitates formation indicates the presence of tannins (Latif et al., 2019).

Flavanoids:
Sodium hydroxide test for qualitative presence: To 5 mL of the aqueous extract filtrate,10 % sodium hydroxide aqueous solution (2 mL)was added.The test solution became yellow.On the addition of dilute HCl, the color changed from yellow to colorless, which was an identification sign for the presence of flavonoids (Latif et al., 2019).

Glycosides:
Liebermann's test: Crude extract from plants mixed with 2mL of chloroform and 2ml of acetic acid.
The mixture was ice-cooled then H2SO4 was added.Change in color from violet to blue to green indicated the presence of glycone, the main nucleus of steroids (Yadav & Agarwala, 2011).

Steroidal compounds:
An equal volume of chloroform was added to the crude extract of the plant.H2SO4(conc)was added along the side of the test tube.The red color appeared in the lower chloroform layer indicated the presence of steroids (Yadav & Agarwala, 2011).

Determination of Total Flavonoids Content Quantitatively:
Standard curve; Crude extract(0.5mL)was dissolved in 0.1ml of 1M potassium acetate and equal volume of10%aluminum chloride added with methanol1.5 ml and 2.8mLof distilled water.The mixture was kept at room temperature for 30 minutes.The optical density of the mixture was recorded spectrophotometrically at 415 nm.To prepare a standard 100 μgmL -1 stock solution, 10mg of rutin was dissolved in 100 mL of methanol.Further dilutions from the 0.2 to 1μgmL -1 were made from the stock solution.The standard curve was prepared by measuring the absorbance of concentrations i.e. 0.2, 0.4, 0.6, 0.8, and 1 μgmL -1 of rutin solution (Amir, Khan, Mujeeb, Ahmad, & Siddiqui, 2011).A Triplicate assay was performed.Results were expressed as μgmL -1 of rutin concentration using the calibration curve: Y = 2.142x-0.1,R 2 = 0.974, where Y was the absorbance, and x was the rutin concentration.To the crude mL of extract (1mL), 3 to 4 drops of 5% neutral ferric chloride solution were added.The dark green color appeared which indicated the presence of phenols (Ben, Woode, Abotsi, & Boakye-Gyasi, 2013).FolinCiocalteu reagent: In 700 ml of distilling water, 25 g of sodium molybdate, 100 g of sodium tungstate, 50 mL of 85% phosphoric acid, and 100 mL of concentrated HCl was added.The mixture was refluxed for 10 hours after adding 150g of lithium sulphate).
2.5.9.Quantitative determination of Total Phenolic Contents: Standard curve; To determine phenolic content, the standard curve of gallic acid was prepared with a concentration of 0.1, 0.2, 0.4, 0.6, 0.8, and 1 μgml -1 .Gallic acid (0.5mL) was dissolved into 5 mL of diluted FolinCiocalteu reagent (1:10 in distilled water) and 4 mL of 1M aqueous Na2CO3.The absorbance was recorded at 765 nm spectrophotometrically after incubating the reaction mixtures for 30 min at room temperature.The assay was performed in triplicate.Results were expressed as equivalents of gallic acid in μgmL -1 using the calibration curve (Amir et al., 2011).

Antioxidant check by DPPH radical scavenging assay:
To determination the free radicals scavenging activity of plant extract, the methanolic solution of DPPH 0.12 mM was mixed with 1mL plant extracts with concentration ranging from 0.02 to 0.5mgmL -1 .DPPH generated free radicals were picked up by antioxidants after 30 min of incubation in dark.Spectrophotometrically absorbance was taken at 517 nm.DPPH scavenging activity  The microbial strains of Gram-negative and gram-positive kept at 4 °C inoculated on agar slant and subcultured at 37 °C for 48 hours on nutrient agar before antibacterial assay.The antibacterial activity was conducted using Nutrient Agar plates (Latif et al., 2019)(figure 2).
Figure 2. Flowsheet for the antibacterial and antioxidant activity of selected plant extracts.

Statistical analysis of data:
The results obtained were expressed as mean values and standard deviation (SD) of three replicates.
The results were analyzed using one-way ANOVA.The level P < 0.05 was considered as the cutoff value for significance.

Bioinformatics proof
Chimera 1.13.1 was used for docking between ligands and microbes receptors (Pettersen et al., 2004).
Phenolic substances were determined by spectrophotometric analysis at 415 nm after constructing a standard curve of rutin (figure 5).Statistical results are shown in Table 2.The plant extracts inhibited the microbial growth against Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and S. aureus which had mean inhibition zone expressed in the table (3).
The minimum inhibitory concentration (MIC) of 2 mg/mL was observed in the plant extracts against KlebsiellaandEscherichia coli, whereas, 5mg/ml against Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella typhi.

Bioinformatic tools
Docking between the important component (murine of peptidoglycan layer) of bacterial cell wall and different phytochemical constituents proved the experimental results of analysis as explained in table 4.  The results obtained revealed that Solanumnigrum, Daturainoxia, and capsicum annum species inhibited four out of five microorganisms evaluated in this research.The methanolic extracts of three selected plants exhibited weak activity against E. coli and P. aeruginosa yet their aqueous extracts could not inhibit Gram-negative bacteria as correlated with the study of root extract of Datura stramonium and Capsicum annum (Niño, Correa, & Mosquera, 2006)which were not active against.aeruginosa and E.coli.DPPH free radicals scavenging activity attained highest 0.6 and 2 mgmL -1 concentration from crude methanolic extracts as compared with the standard (BHT) showing IC50 of 1.0 mgmL -1 .These findings contribute to assessing more bioactive compounds from other families to elucidate important antimicrobial and cytotoxic agents.

Conclusion
The extracts of various plant species of Solanaceae family have a considerable concentration of Phytochemicals.
Moreover, the young leaves of Solanumnigrum, Daturainoxia, and fruit of capsicum annum were comprised of a valuable amount of phenols and flavonoids, which impart them free radical scavenging and antibacterial activity truly ensuring their anticancer role.The insilico assay emphasizes the interaction between protein structures and flavanoids/ alkaloids for antibacterial activity.

Figure 1 .
Figure 1.Standard curve of total flavonoid content

Figure 5 .
Figure 5.Total flavonoids in Solanum nigrum, Daturainoxia, and capsicum annum plants Free radical scavenging of DPPH activity: Methanolic extracts of Daturainoxia, Solanumnigrum, and Capsicum annum were analysed for free radicals scavenging activity introduced by DPPH.The antioxidant properties of plant extracts were analysed, Daturainoxiarepresented classical trend and highest radical scavenging property as depicted in (fig 4).Different concentrations ranging from 0.2 to 2 mg/mL of the plant extracts were analysed to determine their percentage activity.Daturainoxia, Solanumnigrum, and Capsicum annum scored 81.3 %, 66 %, and 53.2% activity in DPPH assay.

Figure 7 .
Figure 7. Docking results of ligands with receptor murine.A) Docking of Murine and Saponin with one hydrogen bond of length 2.47 Å. (B) Murine-Flavone; one hydrogen bond with 1.98 Å bond length.(C) Murine-Phenol; one hydrogen bond with 2.285 Å bond length.(D) Murine-Alkaloid; two hydrogen bonds with 1.96 and 2.56 Å bond lengths.(E) Murine-Steroid; one hydrogen bond with 2.38 Å bond length4.DiscussionQuite a lot of studies have investigated the antioxidant properties of medicinal plants.Mainly biological antioxidants are obtained from plants in the form of phenolic compounds.Among phytochemicals, steroids are known to draw most of the metabolic processes in the body i.e. sex hormones(Yadav & Agarwala, 2011).The basic 15 carbon skeleton of flavonoids is constituent of phenolic derivatives, which are water-soluble and bear colored pigments.Flavonoids are glycosides that slightly differ from the flavan structure as their glyconeshas central location in nucleus(Bashir et al., 2013).They are hydroxylated phenols which are prepared by plants in response to various pathogens.These flavonoids substances complex the soluble proteins with the peptidoglycans layer in the cell wall of bacteria, delivering vitro bactericidal activity(Yadav & Agarwala,