Correlation between Antibacterial Activity and Free-Radical Scavenging: In-Vitro Evaluation of Polar/Non-Polar Extracts from 25 Plants

Objectives: The current study aimed to measure the antioxidant and antibacterial activities of 25 wild Palestinian edible plants, which were subjected to extraction by polar and non-polar solvents. Correlations between free radical scavenging activity and antibacterial activity of the extracts were assessed for both polar and non-polar fractions. Materials: Twenty-five wild edible plant species that are frequently consumed by people in Palestine (mainly in a rural area) were examined. Among them, 10 plant species were among those with the highest mean cultural importance values, according to an ethnobotanical survey that was conducted in the West Bank, Palestine, a few years ago. Method: The protocol of the DPPH assay for testing free-radical scavenging was utilized for determining EC50 values, while microdilution tests were conducted to determine the 50% inhibitory concentration (IC50) of the extracts for the microorganism Staphylococcus mutans. Results and Discussion: Eight extracts (non-polar fractions) were found to possess an antibacterial IC50 of less than 20 ppm, such as Foeniculum vulgare, Salvia palaestinafruticose, Micromeria fruticose, Trigonella foenum-graecum, Cichorium pumilum jacq, Salvia hierosolymitana boiss, Ruta chalepensis, and Chrysanthemum coronarium. The polar fractions possess higher antioxidant activity, while non-polar fraction possess higher antibacterial activity. Looking at all the results together can deceive and lead to the conclusion that there is no correlation between antibacterial activity against S. mutans and free radical scavenging (R2 equals 0.0538). However, in-depth analysis revealed that non-polar plant extracts with an EC50 of free radical scavenging ≤100 ppm have a four-fold order of enrichment toward more activity against S. mutans. These findings are of high importance for screening projects. A four-fold order of enrichment could save plenty of time and many in screening projects. The antibacterial active extracts marked by low-medium free radical scavenging might act through a mechanism of action other than that of highly active, free radical scavenging extracts. Conclusion: The screening of antioxidant and antimicrobial activity performed on 25 selected wild plant extracts revealed a satisfactory free radical scavenging and antimicrobial potential that could be of value in the management of oxidative stress. Further studies are recommended to explore novel and highly active natural antibacterial products.


Introduction
Many current commercial drugs and cosmetics are derived from natural sources. Naturally derived products are inherently better tolerated in the human body as opposed to synthetic chemicals, and, therefore, have a greater chance of being approved as drugs [1,2]. Modern therapeutics derived from natural products, particularly herbal-based drugs, averaged more than 70% of all drugs in use by the

Plant Extraction
The plants were collected from the mountains of the Nablus region and Kabul mountain (north Galilee) during the period May 2018 to April 2019. They were identified and authenticated by Prof. Khaled Sawalha of the Biology Department at Al-Quds University. Three batches from each species were collected and mixed together to prepare the representative material of each plant. The specimens were washed with distilled water and dried in the shade for one month. Then, they were ground up and packed in the tubes of Soxhlet (Corning, Inc., NY, USA). In a round-bottomed flask, a mixture of solvents composed of 60 mL of water, 30 mL of ethanol, 30 mL of ethyl acetate, and 30 mL of hexane was introduced. The aim of using a mixture of solvents is maximizing natural products extraction into polar and non-polar layers. After the extraction was completed (following 3 cycles), the polar and non-polar phases were separated with a separation funnel. Both the polar and non-polar extracts were filtered by passing the solutions through 0.2-µm disposable filters. The solvents from each extract were evaporated in an oven under reduced pressure. The polar extracts (free of solvents) were dissolved in water, while the non-polar extracts were dissolved in dimethyl sulfoxide (DMSO) both at a concentration of 20,000 ppm. The prepared solutions were subjected to free radical scavenging tests using the DPPH assay. Microdilution tests were also conducted to determine the IC 50 s of the extracts against S. mutans.

Free Radical Scavenging Activity
There are several assays that are used to determine antioxidant activity of plant extracts. The most used one is based on free radical scavenging of the antioxidant to the stable chromogenic radical scavenge DPPH (2,2-diphenyl-1-picryl hydrazyl). The reduction of DPPH by the hydrogen donor antioxidants cause decolorization of the dark purple color of DPPH and, as a result, the absorption decreases. The method is simple, rapid, accurate, and inexpensive. Currently, more than 17,000 articles were cited using this simple and straightforward method [34]. The method was first invented by Blois [35] and then modified by many investigators [36]. In the current study, the free radical scavenging activity of the various extracts was measured by microdiluting the DPPH assay, with some modifications. The microdilution of the DPPH assay was performed using two-fold serial dilution in ethanol. The tests were carried out in 96-well, flat-bottomed microtitration plates. The absorbance of the solution was measured at 517 nm and converted into a percentage of free radical scavenging activity using Equation (1).
where the A sample is the absorbance of the mixture (plant extract and DPPH), A blank _ 1 is the absorbance of the plant extract, A control is the absorbance of the ethanolic solution of DPPH, and A blank _ 2 is the absorbance of the blank (solvent). Gallic acid was used as a positive control. The free-radical scavenging was expressed in terms of EC 50 (the amount of antioxidant necessary to decrease the initial DPPH absorbance by 50%). The EC 50 value for each plant extract was determined by extracting the value from the equation for the linear part of the graph. We substitute 50% for the y value while calculating the concentration value of the x-axis.

Antibacterial Activity
The microdilution test was used to determine the 50% inhibitory concentration (IC 50 ) of the different extracted plant samples. A broth microdilution assay was performed using two-fold serial dilution in brain heart infusion (BHI) broth. The test was carried out in 96-well, flat-bottomed microtitration plates. The cell suspension was prepared in BHI broth with an optical density equivalent to the 0.5 McFarland standard and diluted 1:100 in BHI broth to obtain a final concentration of 5 × 10 5 clone-forming units per milliliter (CFU/mL). Controls of broth only and broth with bacteria without any of the antibacterial agents were also included in each plate. One hundred µL of antibacterial agent was put in the first microplate well and serially diluted in BHI broth. One hundred µL, corresponding to 5 × 10 5 CFU/mL, was added to all the wells. The plates were incubated at 37 • C for 24 h. For more details regarding the cultivation condition, see our recent publications [1,37]. Tetracycline was used as a positive control for S. mutans. The IC 50 was defined as the concentration able to inhibit the growth of bacteria in the quadruplicate wells. The absorbance of the solution was measured at 620 nm and converted into a percentage of bacterial growth inhibition, using the Equation (2).
where A sample_24 is the absorbance of the mixture (plant extract and bacteria) after 24 h, A sample_0 is the absorbance of the mixture (plant extract and bacteria) at time 0, A control_24 is the absorbance of the untreated bacteria wells after 24 h, and A control_0 is the absorbance of the untreated bacteria wells at time 0. The antibacterial IC 50 for each plant extract was determined by extracting the value from the equation of the linear part of the graph. The negative control was DMSO solvent and, as reported in previous publications [1,37], its minimum inhibitory concentration (MIC) value is 25% (v/v).

Model Assessments
Parameters such as Matthew's correlation coefficient (MCC) [38], accuracy, precision, enrichment factors, and the area under the ROC curve (AUC) were used to assess the quality of the antibacterial/ free radical scavenging correlation models.
Equation (3): Equation (4): Accuracy Equation (5): Precision Precision = P/(P + P f ) Equation (6): Enrichment factor where P, N, P f , and N f are the numbers of true positive, true negative, false positive, and false negative predictions, respectively. T FRS is the percentage of actives when using the free radical scavenging threshold criterion and T RS is the percentage of actives by random selection.

Statistical Analysis
All data are presented as the average of three to four experiments, and error bars display the standard errors of deviations. All statistical analyses were conducted using Microsoft EXCEL 2013 with its Data Analysis add-in (v16.0, Microsoft, Redmond, WA, USA). The dose-response curves for scavenging activity were plotted based on the percentage of scavenging and the concentration. The EC 50 values for free radical scavenging were determined with linear regression analysis. Reliability lowers with a decrease in the R 2 value. Differences between the groups were evaluated by applying a one-way analysis of variance (ANOVA). A p-value of less than 0.05 is considered statistically significant.

Results and Discussion
Twenty-five wild edible plants were studied for their free radical scavenging and antibacterial activity against S. mutans. Antibacterial potential was first verified by screening all the extracts (polar and non-polar fractions) for their activity, using one concentration of 250 ppm. The polar fractions were less active than the non-polar fractions. Column 3 of Table 1 depicts the EC 50 of the free radical scavenging and the percentage of inhibition against S. mutans at a 5000-ppm concentration of the polar fraction. The extracts that exhibited a percentage of bacterial inhibition above 60% were tested in the second round at lower concentrations in a dose-response manner to extract their IC 50 values. The antibacterial IC 50 values of the polar fraction of the three plants Ruta chalepensis, Rumex patientia L., and Portulaca oleracea L. are 1750 ppm, 3925 ppm, and 4133 ppm, respectively. Column 4 of Table 1 depicts the EC 50 of the free radical scavenging and the IC 50 against S. mutans. Eight extracts (non-polar fractions) were found to possess relatively high antibacterial IC 50 values of less than 20 ppm such as Foeniculum vulgare, Salvia palaestina, Micromeria fruticose, Trigonella foenum-graecum, Cichorium pumilum jacq, Salvia hierosolymitana boiss, Ruta chalepensis, and Chrysanthemum coronarium.
Rules-based analysis using Matthew's correlation coefficient (MCC) scores and enrichment factors as criteria for evaluating the models' efficiency revealed that the plant extracts whose EC 50 for free radical scavenging <100 ppm showed some degree of enrichment toward being more active as an antibacterial agent against S. mutans (see Table 2). The values for the enrichment factor, the MCC, accuracy, and precision were 4.1, 0.217, 0.68, and 0.5, respectively. These findings are of high importance for screening projects. A four-fold order of enrichment toward more activity against S. mutans for the extracts with an EC 50 of free radical scavenging ≤100 ppm could save plenty of time and could save many in screening projects. Considering all the results can lead to the conclusion that there is no correlation between antibacterial activity against S. mutans and free radical scavenging (as shown in Figure 1, R 2 equals 0.0538), and also for the eight most active non-polar plant extracts (as shown in Figure 2, R 2 equals 0.0469). Three of the eight active plant extracts (Salvia palaestina, Micromeria fruticose, and Salvia hierosolymitana boiss) show relatively high free radical scavenging, and four others (Foeniculum vulgare, Cichorium pumilum jacq, Ruta chalepensis, and Chrysanthemum coronarium) show medium free radical scavenging, while the remaining extract (Trigonella foenum-graecum) shows relatively low free radical scavenging. The results show that differences in antibacterial activity among the non-polar extracts cannot always be attributed to antioxidant levels but could be associated with inhibitory effects via other mechanisms of action [39] (inhibition or regulation of enzymes involved in cell wall biosynthesis, nucleic acid metabolism and repair, protein synthesis, or disruption of the membrane structure). regulation of enzymes involved in cell wall biosynthesis, nucleic acid metabolism and repair, protein synthesis, or disruption of the membrane structure).    Figure 4 shows the receiver operating characteristic (ROC) plot for the antibacterial/ free radical scavenging correlation model. The area under the curve (AUC) that was attained for the current model, as shown in Figure 4, is 0.661, which means that the model is very poor. This indicates a very weak correlation between antibacterial and free radical scavenging. The enrichment plot, which is shown in Figure 3, illustrates how quickly antibacterial extracts of plants can be identified when they are sorted based on their free radical scavenging activity. In-depth analysis revealed that the shape of the figure fits well with the conclusions drawn     Figure 4 shows the receiver operating characteristic (ROC) plot for the antibacterial/ free radical scavenging correlation model. The area under the curve (AUC) that was attained for the current model, as shown in Figure 4, is 0.661, which means that the model is very poor. This indicates a very weak correlation between antibacterial and free radical scavenging. The enrichment plot, which is shown in Figure 3, illustrates how quickly antibacterial extracts of plants can be identified when they are sorted based on their free radical scavenging activity. In-depth analysis revealed that the shape of the figure fits well with the conclusions drawn   Figure 4 shows the receiver operating characteristic (ROC) plot for the antibacterial/ free radical scavenging correlation model. The area under the curve (AUC) that was attained for the current model, as shown in Figure 4, is 0.661, which means that the model is very poor. This indicates a very weak correlation between antibacterial and free radical scavenging. The enrichment plot, which is shown in Figure 3, illustrates how quickly antibacterial extracts of plants can be identified when they are sorted based on their free radical scavenging activity. In-depth analysis revealed that the shape of the figure fits well with the conclusions drawn from the  Table 2, which shows that non-polar plant extracts with an EC 50 of free radical scavenging <100 ppm have a four-fold order of enrichment toward more activity against S. mutans. from the detailed analysis of Table 2, which shows that non-polar plant extracts with an EC50 of free radical scavenging <100 ppm have a four-fold order of enrichment toward more activity against S. mutans.   from the detailed analysis of Table 2, which shows that non-polar plant extracts with an EC50 of free radical scavenging <100 ppm have a four-fold order of enrichment toward more activity against S. mutans.    When the eight active plants were searched through electronic databases, including PubMed, we found a huge number of relevant reports documenting their antibacterial potential on different statins, except for Salvia hierosolymitana. For example, the antimicrobial activity of Foeniculum vulgare has been extensively reviewed [40]. The antibacterial activity of its essential oil against S. mutans showed an MIC and an MBC of 8.4 and 14.9 ppm, respectively. Salvia palaestina was also recently investigated by us for its antioxidant and antimicrobial potential [41]. The antimicrobial activity was examined by the disc diffusion method, and 5 µL (100%) of the essential oil was found to exceed the activity of gentamicin in the case of S. aureus, while it was nearly as effective as gentamicin against E. coli. Furthermore, according to both SEM and crystal-violet staining data, the essential oil of Micromeria fruticosa was found to exert an inhibitory effect on S. mutans biofilm formation [42]. Trigonella foenum-graecum (fenugreek) was also found to be effective in amounts of 20 mm and 19 mm against E. coli and Staphylococcus, respectively [43]. Cichorium pumilum, which is widely used as a traditional medicinal herb, showed antibacterial activity in vitro with agar disc diffusion and agar well diffusion methods against 10 different bacterial species [44]. The antimicrobial activity of Ruta chalepensis L. was tested against four pathogenic bacteria-Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa-and the findings indicate possible applications for the treatment of various infectious and noninfectious diseases [45]. The antibacterial effects of essential oil of Chrysanthemum coronarium demonstrated pronounced activity against gram-positive strains of bacteria: Bacillus subtilis (19 mm), Staphylococcus aureus (20 mm), Staphylococcus epidermidis (18 mm), Escherichia coli (9 mm), and Pseudomonas aeruginosa (12 mm), which were assessed by measuring the zone of inhibition [46]. Another unique aspect of this study is the scanning of all the 25 plant extracts at the same lab by using the same procedures for testing their antioxidant and antibacterial activities.

Conclusions
For this study, free radical scavenging was assessed by a DPPH assay, while antibacterial activity was measured by a microdilution assay. Eight wild plant extracts (non-polar fractions) were found to possess antibacterial IC 50 values of less than 20 ppm, including Foeniculum vulgare, Salvia palaestina, Micromeria fruticose, Trigonella foenum-graecum, Cichorium pumilum jacq, Salvia hierosolymitana boiss, Ruta chalepensis, and Chrysanthemum coronarium. In addition, we raise a question regarding the correlation between free radical scavenging and antibacterial activity. Answering this question might be helpful for making the screening process more efficient. Looking at all the results together can deceive and lead to the conclusion that there is no correlation between antibacterial activity against S. mutans and free radical scavenging (R 2 equals 0.0538). However, an in-depth analysis of the results revealed that the extracts of plants that exhibited an EC 50 of free radical scavenging of ≤100 ppm showed some degree of enrichment (four orders of magnitude) toward more antibacterial activity. These findings are of high importance for screening projects. A four-fold order of enrichment could save plenty of time and many in screening projects. The antibacterial activity of the extracts could be attributed to various mechanisms of action. The highly active free radical scavenging extracts might follow one mechanism of action, while the extracts that are marked by low-medium free radical scavenging might act through another mechanism of action. This hypothesis could explain the enrichment that exist in extracts having EC 50 of free radical scavenging of ≤100 ppm.
Our next study will aim to discover novel and highly active natural antibacterial products, using a novel in-house bioassay guided fractionation approach [1,28,31,37] and in-silico based modeling techniques [29,[47][48][49][50] to identify natural products that exhibit antibacterial activity against S. mutans.