The Influence of Hypericum perforatum L. Addition to Wheat Cookies on Their Antioxidant, Anti-Metabolic Syndrome, and Antimicrobial Properties

The aim of this study was to characterize wheat cookies enriched with 0.5% and 1.0% of Hypericum perforatum L. (St. John’s wort, SJW) and determine their pro-health properties in vitro after hydrolysis in simulated gastrointestinal conditions. The results indicated that 1.0 SJW was characterized by the highest content of polyphenols, flavonoids, and phenolic acids (2.32 mg mL−1, 4.93 µg mL−1, and 12.35 µg mL−1, respectively). The enriching cookies had no effect on water absorption capacity (WAC) and oil absorption capacity (OAC). After in vitro hydrolysis, the highest peptide content was noted in 1.0 SJW (0.52 mg mL−1), and the bioactive compounds were characterized by high potential bioaccessibility (PAC), but poor bioavailability (PAV). The addition of SJW increased the ACE, α-amylase, and LOX inhibitory effect, but reduced the inhibition of pancreatic lipase. The highest antioxidant activity was noted for 1.0 SJW. The results showed that only 0.5 SJW and 1.0 SJW had slight antimicrobial activity against E. coli ATCC 25922 and B. cereus ATCC 14579 with MIC = 12.5 mg mL−1. Fractions with molecular mass <3.0 kDa were characterized with the highest p-coumaric acid content. The results show that SJW cookies had a higher content of bioactive compounds and more potent anti-metabolic syndrome effects.


Introduction
In recent years, there has been a rapid development of the food industry, which has brought some benefits as well as negative effects. Nowadays, the pace of life is frighteningly fast, and people are focused on work and improvement of their career, which results in a lack of time to prepare meals. Consequently, there is a demand for takeaway, ready-to-eat, and pre-prepared meals, which are often processed food [1]. However, it is observed that nutritional decisions have an influence on human health and an increase in the incidence of chronic diseases associated with the development of civilization such as metabolic syndrome, type 2 diabetes, polycystic ovary syndrome (PCOS), tumors, or autoimmune disease [2,3]. Furthermore, obesity is an effect of imbalance between the amount of consumed calories and physical activity [4].
Metabolic syndrome (MS) is a chronic disease comprising insulin resistance, hypertension, obesity, large waist circumference (according to the International Diabetes Foundation: amounts of the herb [9]. Moreover, SJW has been described as a plant that may be potentially used in the treatment of many other diseases like AIDS or cancer, and exhibits antioxidant, antidiabetic, or anti-inflammatory properties [18].
The aim of the study was to assess the impact of Hypericum perforatum on the antioxidant activity, enzyme inhibitory effect, and antimicrobial properties of wheat flour cookies supplemented with St. John's wort.

Plant Material
Dry St. John's wort (Hypericum perforatum L.) was purchased at a local market in Istanbul (Turkey).

Procedure for Preparation of Cookies
The cookies were prepared as in Złotek [19] with slight modification: the wheat flour was replaced with the St. John's wort (SJW) herb at 0.5 and 1 g/100 g (0.5 SJW and 1.0 SJW, respectively). Cookies without St. John's wort were the control samples (control). All cookies were dried and used for further analysis.

Preparation of Extracts
The cookies were ground in a laboratory grinder (SM-450C, MRC, Warsaw, Poland). The water extracts were prepared according to the method described by Jakubczyk et al. [20].

Polyphenol Content Assay 2.4.1. Total Phenolic Content (TPC)
Total phenolic content was determined in water extracts and fractions with molecular mass <3.0 kDa using Folin-Ciocalteau reagent [21]. TPC was calculated as gallic acid equivalent (GAE) µg per g of dry weight (DW).

Flavonoid Content Assay (FCA)
FCA was determined with the method described by Lamaison and Carnet [22] and calculated as quercetin equivalent (QE) in µg per g of dry weight (DW).

Phenolic Acid Content Assay
Estimation of total phenolic acids was carried out with the Arnov method [23] and expressed as caffeic acid equivalent (CAE) in µg per g of dry weight (DW).

Reducing Sugar Content
The reducing sugar content before hydrolysis was determined using the DNS method [24].

Peptide Content Assay
The assay was performed using the Adler-Nissen [25] method with L-leucine as a standard. WAC of the samples was determined according to the method described by Khattab and Arntfield [26] with modification described by Jakubczyk et al. [20].

Oil Absorption Capacity (OAC)
OAC was determined as in Khattab and Arntfield [26] with modification described by Jakubczyk et al. [20].

In Vitro Hydrolysis
In vitro digestion of the cookies was carried out with the method described by Durak et al. [27].

Preparation of Fractions with Molecular Mass <3.0 kDa
Fractions with molecular mass <3.0 kDa were obtained according to the method described by Jakubczyk et al. [20].

Potential Bioaccessibility (PAC) and Bioavailability (PAV) of Bioactive Compounds Obtained from Cookies
The theoretical calculation of the nutritional potential was based on the index described by Gawlik-Dziki et al. [28]. The bioaccessibility index (PAC), which is an indicator of the bioaccessibility of bioactive compounds, is expressed as: PAC = Cph/Cpb Cph-bioactive compound content in the hydrolyzate Cpb-bioactive compound content in the sample before hydrolysis The peptide bioavailability index (PAV), which is an indicator of the bioavailability of bioactive compounds, is expressed as: PAV = Cpa/Cph Cpa-bioactive compound content after the absorption process Cph-bioactive compound content in the hydrolyzate 2.10. Antioxidant Activity 2.10.1. ABTS •+ Antiradical activity against ABTS •+ was determined with the method described by Re et al. [29]. All assays were performed in triplicate. The EC 50 (mg mL −1 ) value was defined as an effective concentration of the sample that is required to scavenge 50% of radical activity.

DPPH •
Antiradical activity against DPPH • was determined with the method proposed by Brand-Williams, Cuvelier, and Berset [30]. All assays were performed in triplicate. The EC 50 (mg mL −1 ) value was defined as an effective concentration of the sample required to scavenge 50% of radical activity.

Fe 2+ Chelating Activity
The method developed by Decker and Welch [31] was used to determine the ability of samples to chelate ferrous ion (II). All assays were performed in triplicate.
2.11. Determination of Enzyme Inhibitory Activity 2.11.1. Angiotensin Converting Enzyme (ACE) Inhibitory Activity ACE inhibitory activity was measured by spectrophotometry with o-phtaldialdehyde as in Jakubczyk et al. [32]. The EC 50 (mg mL −1 ) value, defined as the concentration of the sample that inhibits 50% of ACE activity, was determined by measuring the ACE inhibitory activity and peptide contents of each sample.

Pancreatic Lipase Inhibitory Activity
Lipase inhibitory activity was determined with the method described by Jakubczyk et al. [32].

Lipoxidase (LOX) Inhibitory Activity
The LOX inhibitory assay was carried out with the method described by Szymanowska et al. [33].

Antimicrobial Properties
The samples were tested against bacteria Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29737, Listeria monocytogenes ATCC BBA-2660, Bacillus cereus ATCC 14579, and Salmonella enteritidis ATCC 4931 as well as yeast Candida albicans ATCC 90028. These strains were obtained from the American Type Culture Collection (ATCC, distributors: LGC Standards, Łomianki, Poland) and stored at 4 • C. All strains were cultured at 37 • C on Nutrient Broth (NB) medium.

Biotoxicity Assay Using the Resazurin Reduction Method
The biotoxicity against bacteria and yeast was assessed using a resazurine reduction assay as in Jakubczyk et al. [36].  Table S1). The LC-MS/MS system was controlled using Agilent MassHunter software, which was also used for data processing.

Statistical Analysis
All determinations were performed in triplicate. Statistical analysis was performed using STATISTICA 13.3. software for mean comparison using ANOVA with Tukey's post-hoc honestly significant difference (HSD) test at the significance level α = 0.05.

Characteristics of Cookie Extracts
In general, the addition of St. John's wort to the wheat cookies had an impact on the content of phenolic compounds, but had no statistically significant effect on water and oil absorption ( Table 1). Sample 1.0 SJW had the highest level of total phenolics, flavonoids, and phenolic acid content (2.32 mg g −1 , 4.93 mg g −1 , and 12.95 µg g −1 , respectively). There were differences between the 0.5 SJW and 1.0 SJW samples in the content of flavonoids and phenolic acids, but the difference between the total phenolic amount was statistically insignificant. It should be noted that the addition of SJW to the cookies had no effect on the WAC and OAC values.

Peptide Content during Hydrolysis
The cookies were subjected to in vitro digestion in gastrointestinal conditions. The peptide content was measured and shown in Figure 1. The α-amylase activity resulted in an increase in the level of peptides in each sample. The 1.0 SJW sample was characterized by the highest level of peptides in each step of hydrolysis compared to the other samples, but only α-amylase and pepsin action exerted a significant effect. The highest peptide content was determined for the 1.0 SJW sample after pancreatin hydrolysis (0.52 mg mL −1 ) but this value was not statistically significant. The differences between the peptide content in the control and the 0.5 SJW sample after α-amylase and pepsin hydrolysis were not statistically significant.

Bioactive Compounds in Hydrolyzates and Fractions with Molecular Mass <3.0 kDa
After hydrolysis, the samples were subjected to the procedure of obtaining fractions with molecular mass <3.0 kDa. Table 2 shows that the polyphenol content was similar in each sample of the hydrolyzates and fractions separately. It should be noted that the fractions contained a statistically significantly higher amount of this bioactive compound. The highest level of flavonoids was observed in the 1.0 SJW sample (29.06 µg g −1 ). The fractions were characterized by a lower content of phenolic acids than the hydrolyzates. In turn, the St. John's wort had a statistically significant effect addition in the group of hydrolyzates. The same was observed for the values of the peptide content. The content of reducing sugar was statistically the same in the hydrolyzates and fractions.

Bioactive Compounds in Hydrolyzates and Fractions with Molecular Mass <3.0 kDa
After hydrolysis, the samples were subjected to the procedure of obtaining fractions with molecular mass <3.0 kDa. Table 2 shows that the polyphenol content was similar in each sample of the hydrolyzates and fractions separately. It should be noted that the fractions contained a statistically significantly higher amount of this bioactive compound. The highest level of flavonoids was observed in the 1.0 SJW sample (29.06 µg g −1 ). The fractions were characterized by a lower content of phenolic acids than the hydrolyzates. In turn, the St. John's wort had a statistically significant effect addition in the group of hydrolyzates. The same was observed for the values of the peptide content. The content of reducing sugar was statistically the same in the hydrolyzates and fractions.

PAC and PAV Indexes of Bioactive Compounds
Besides the bioactive compound content, we established their potential bioaccessibility and bioavailability factors ( Table 3). The compounds exhibited good bioaccessibility in vitro including reducing sugar. The values for all of the samples were higher than 1, and the highest factor for polyphenols and phenolic acids was noted in the 0.5 SJW sample (3.34 and 1.47, respectively) and for flavonoids in the control sample (12.01). The PAC indexes for peptides and reducing sugar were similar (around 4.00 and 0.46, respectively). However, no sample exhibited bioavailability since this factor was lower than one in all cases. The highest PAV index for polyphenols, flavonoids, and reducing sugar was noted for the 0.5 SJW sample (0.83, 0.13, and 0.99, respectively). Moreover, the highest PAV index for phenolic acids and peptides was determined for the control sample (0.29 and 0.72, respectively).  Table 4, the addition of St. John's wort resulted in higher antioxidant activity against ABTS of fractions with molecular mass <3.0 kDa, but it was not statistically significant. Nevertheless, the inactivation capability was lower in the hydrolyzates of the same sample. In the group of fractions, the lowest significant EC 50 value of the antioxidant activity against DPPH was noted for the control sample = 0.12 mg mL −1 , but it was not statistically significant compared to the same hydrolyzate sample. The hydrolyzates from the enriched cookies were shown to have an impact on the antioxidant power compared to the control sample. The fractions with molecular mass <3.0 kDa had higher antioxidant properties than the hydrolyzates for the same sample. The Fe 2+ chelation ability was almost identical for each sample of the hydrolyzates and fractions. The highest antioxidant activity was observed for the control and 0.5 SJW samples (87.23% and 87.09%, respectively), but these values were not statistically significant compared to the rest of the results.

Assay of Inhibition of Enzymes Involved in Metabolic Syndrome Pathogenesis
The impact of the hydrolyzates and fractions with molecular mass <3.0 kDa on enzymes involved in metabolic syndrome pathogenesis was investigated. Clearly, the highest influence on enzyme inhibition was exhibited by the fractions, especially in the case of α-amylase. As shown by the data in Table 5, the 1.0 SJW hydrolyzate samples from cookies enriched with St. John's wort exhibited increased activity against ACE, LOX, and αamylase function (EC 50 1.24 mg mL −1 , 1.03 mg mL −1 , and 0.05 mg mL −1 , respectively), but the value of ACE inhibition was not statistically significant. The lowest effect of the St. John's wort addition was observed for lipase activity.

Antimicrobial Effect
The   Table 6. It should be noted that not all standards were identified in the tested samples. The addition of St. John's wort exerted an effect on the content of most of the identified compounds. The highest concentration was noted for p-coumaric acid in the 1.0 SJW sample, but gallic acid and salicylic acid were determined under a lower limit of quantitation (LLOQ = 0.5 µg g −1 ).

Phenolic Compounds in Fractions with Molecular Mass <3.0 kDa Identified with the LC-MS/MS Method
Phenolic compounds identified by the LC-MS/MS method are shown in Table 6. It should be noted that not all standards were identified in the tested samples. The addition of St. John's wort exerted an effect on the content of most of the identified compounds. The highest concentration was noted for p-coumaric acid in the 1.0 SJW sample, but gallic acid and salicylic acid were determined under a lower limit of quantitation (LLOQ = 0.5 µg g −1 ).

Discussion
For the last few years, food has been considered by consumers not only as a source of nutritional compounds, but also as a source of bioactive compounds that may provide various benefits to human health. Due to the pandemic situation of SARS-CoV-2 and COVID-19 disease, people spend more time at home, have low physical activity, and undergo more stress. For this reason, more attention should be paid to nutrition, which should contain compounds with antioxidant activity, preventing the occurrence of diseases such as hypertension, obesity, or diabetes 2.
Food enrichment is one of the methods of increasing the pro-health quality and compound quality of products. This process is applied to many types of food products. The most commonly enriched food is bread [38][39][40], pasta [41][42][43] or snacks such as wafers [20], crackers [44], and cookies [19]. Food enrichment changes the texture, flavor, or aroma of products and alters the technological properties (e.g., baking value or storage conditions). Moreover, it may cause the appearance of new features that were not present in the product before. The additives represent various types of ingredients: waste products, flour, oil, fruit, vegetables, and herbs.
Herbs not only improve the taste and aroma of products but may also have several health properties (e.g., antioxidant, anticancer, anti-inflammatory, or antimicrobial activity) and are applied in the production of many cereal products. The aim of our study was to investigate the influence of Hypericum perforatum L., known as St. John's wort (SJW), on the bioactive compounds and biological activity of wheat cookies. This herb is characterized by a high content of bioactive compounds (e.g., polyphenols, flavonoids, biflavones, phenolic acids, and naphthodianthrones). The plant is used as an alternative method primarily in the treatment of depression as well as rheumatism, gastroenteritis, headache, and neuralgias. The exact mechanism of action is still unknown, but there are studies that have indicated the role of the neurotransmitter inhibitory profile and potential anti-inflammatory and antioxidant effects, suggesting its role in pain treatment [45].
The research material was three types of cookies: control-without the addition of SJW; 0.5 SJW-cookies with 0.5 of St. John's wort; and 1.0 SJW-cookies with 1% of St. John's wort. Since SJW is characterized by high polyphenol content, the total polyphenols, flavonoids, and phenolic acids in extracts obtained from the cookies were determined ( Table 1). As expected, the addition of SJW to the cookies increased the levels of bioactive compounds. It should be noted that the total phenolic content in 0.5 SJW and 1.0 SJW was higher than that in the control sample, but the difference between 0.5 SJW and 1.0 SJW was not statistically significant. The present results are in agreement with the findings reported by Złotek [19], where the total phenolic content in cakes enriched with 1% and 2% of basil was higher than in the control sample, but the difference between C1% and C2% was not statistically significant. In turn, the content of flavonoid and phenolic acid was not statistically significantly higher in the control and 0.5 SJW samples. The addition of St. John's wort increased the content of these bioactive compounds only in the case of 1.0 SJW. This indicates that the food matrix has an influence on the extraction of bioactive compounds [46]. It should be noted that the enriched cookies did not have different WAC and OAC values to the control sample. WAC is an economically important factor for the food industry as loss of moisture adversely affects the yield and quality of products [47]. Hence, some products, especially meat, should be characterized by a high WAC value. However, this factor for cereal products should not be as high, and water absorption levels are often in the range of 60-62% in the standard white bread formula, 80-90% in the artisan-type Ciabatta formula, and 50-54% in the cookie formula [48]. Wheat flour is characterized by 75% WAC [49]. As shown in Table 1, the WAC value of all samples was around 100%, and this is not a high value compared with products enriched with other additives. In our previous research, wheat wafers were enriched with millet flour and the WAC value was above 600% [20]. Another important functional property of cereal products is the absorption capacity (OAC), which contributes to enhancing mouth feel while retaining the flavor of the food product. The ability of food ingredients to bind with oil makes them useful in food production with optimal oil absorption. OAC facilitates enhancement of the flavor and mouth feel when used in food preparation [48]. In our study, the addition of St. John's wort to the cookies had no statistical effect on the increase in this parameter. All samples were characterized by around 160% OAC (Table 1), which indicates that this factor may influence flavor formulation.
All samples were hydrolyzed in vitro in gastrointestinal condition and, afterward, fractions with molecular mass <3.0 kDa were obtained from the hydrolyzates. This fraction potentially contains compounds that can pass through the lumen of the small intestine [50]. In each step of the hydrolysis, the content of peptides was determined (Figure 1). During the hydrolysis, α-amylase was used as the first active enzyme in the gastrointestinal tract. This enzyme hydrolyzes carbohydrates, but the results of our study indicated increased peptide content in the sample after action of this enzyme. This process was also observed in our previous study [20,51,52], which indicated that proteins are combined with polysaccharides and may release protein or peptides when exposed to this enzyme. The results of the present study demonstrated that the addition of SJW to the cookies induced no significant differences in the peptide content, which was noted only between 0.5 SJW and 1.0 SJW after the use of α-amylase and pepsin. The highest peptide content was noted for hydrolyzates obtained from 1.0 SJW after pancreatic hydrolysis (0.52 mg mL −1 ), but the values after the last step of hydrolysis of all samples were not statistically significant.
The content of bioactive compounds was determined in all hydrolyzates and fractions with molecular mass <3.0 kDa. The results show ( Table 2) that all hydrolyzates were characterized by a higher content of bioactive compounds than the fractions. Statistical significance was noted for flavonoids in the 1.0 SJW sample and phenolic acids and peptides in the 0.5 SJW and 1.0 SJW samples. These results indicate that the addition of St. John's wort to cookies had no significant influence on the release of bioactive compounds from the food matrix. Similar results were obtained from the fraction where only reducing sugar content was different in 1.0 SJW from that in the other samples. Besides the content of bioactive compounds in the food matrix, their potential bioaccessibility (PAC) and bioavailability (PAV) factors are very important. All PAC factors were significantly higher than 1, which indicated high potential bioaccessibility of the bioactive compounds (Table  3). These results correspond well with those described byŚwieca et al. [53], where the PAC index for wheat bread enriched with green coffee was also higher than 1. The PAC indexes for all bioactive compounds (except for flavonoids) in the enriched cookies were higher than in the control sample. This suggests that the addition of St. John's wort to cookies may indicate the occurrence of phenolic-cookie matrix interactions. This process was also described for enriched bread [54]. On the other hand, the study results showed that the bioactive compounds obtained from cookies enriched with St. John's wort were characterized by low potential bioavailability. For all compounds, the PAV index was lower than 1, but the 0.5% addition of herbs induced an increase in the potential bioavailability of polyphenols, flavonoids, and reducing sugar (Table 3).
St. John's wort has been recognized as a source of bioactive compounds with antioxidant, antidepressant, anti-apoptotic, anti-obesity, antimicrobial, antiviral, antioxidant, anti-inflammatory, and enzyme inhibitory effects [55]. The exact mechanism of action is still not understood. Nevertheless, St. John's wort components have been described to exert inhibitory on hydrolases such as acetylcholinesterase, α-glucosidase, and α-amylase [56]. For this reason, St. John's wort was chosen as an additive to the formula of cookies. As shown by the results (Table 4), the fractions with molecular mass <3.0 kDa were characterized by higher antioxidant effects than the hydrolyzates. The strongest antiradical effect was noted for sample 1.0 SJW, which indicates that enrichment of cookies with St. John's wort is an effective method to obtain a product that may be used for its therapeutic diet effect. Excessive concentrations of free radicals and weakened defense mechanisms against them can be the cause of many diseases (e.g., cardiovascular diseases, obesity, diabetes, and inflammation processes). These disorders are also a risk factor of the development of metabolic syndrome, which is considered as the leading cause of death [57]. It is believed that the disease will be even more prevalent due to the epidemic situation. An incorrect lifestyle including inappropriate diet, low physical activity, or stress plays a key role in the development of this disease. At the molecular basis, there is the activity of some enzymes, especially hydrolases such as the angiotensin converting enzyme (ACE), which may cause hypertension and cardiovascular diseases, pancreatic lipase that hydrolyzes about 70% of fat from food products in the human gastrointestinal tract, and amylases that hydrolyze polysaccharides into small molecules. Moreover, the inflammation process may generate free radicals and may contribute to the development of obesity. One of the therapeutic methods for reducing the development of metabolic syndrome is the inhibition of the activity of some enzymes. In the present study, we investigated the inhibitory effects of hydrolyzates and fractions with molecular mass <3.0 kDa on their activity. As shown by the present results (Table 5), the addition of St. John's wort increased the inhibitory effect of the product. The highest anti-metabolic effect was noted for the enriched samples. Only the lipase inhibitory activity was not stronger than that of the control sample. The hydrolyzates were characterized by a significantly lower effect than the fractions with molecular mass <3.0 kDa, which may indicate potential bioaccessibility. It should be noted that the reducing sugar content, which may contribute to an increase in postprandial glycemia, was the same in the hydrolyzates and the fractions. In turn, the α-amylase inhibitory effect of the fraction was significantly higher than that of the hydrolyzates. This suggests that the obtained products can be used for a therapeutic diet effect in diabetes.
Foodborne pathogens are responsible for foodborne infections with significant effects on human health. Most food poisoning reports are associated with bacterial contamination, especially by Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus cereus [58]. Consequently, there is a growing interest in functional food ingredients of natural origin, which may affect health in addition to their nutritional value. Our study showed slight but significant antimicrobial activity of cookies enriched with 12.5 mg mL −1 of St. John's wort against E. coli and B. cereus. Avato et al. [59] reported that St. John's wort ethanolic extract inhibited only the growth of Gram-positive bacteria B. subtilis and B. cereus. The authors indicated hypercin and hyperforin as the main antibacterial agents [59]. However, other authors detected antimicrobial activity of H. perforatum not only against E. coli and B. cereus, but also against Shigella dysenteriae, Salmonella typhi, Staphylococcus aureus, Streptococcus mutans, and Enterococcus faecalis [60][61][62].
The identification by the LC-MS/MS method determined that the addition of St. John's wort into wheat cookies preparation had an influence on the content of phenolic compound in fraction with a molecular mass <3.0 kDa. As shown by the data in Table 6, phenolic compounds in samples were gallic acid, chlorogenic acid, 3,4-dihydroxybenzoic acid, rutin, p-coumaric acid, ferulic acid, and salicylic acid. The results corresponded well with that described by Silwa et al. [63] where these compounds were also determined. The highest content of phenolic compounds was noted in the 1.0 SJW sample. It should be noted that the addition of 1.0% of St. John's wort decreased potential bioaccessibility of bioactive compounds such as phenols.

Conclusions
Food enrichment is one of the effective methods of increasing the pro-health potential of products. This process is not only aimed at increasing the pro-health value of food, but can also influence the taste, smell, and texture. The study indicated that cookies enriched with St. John's wort had a higher content of bioactive compounds and antioxidant and anti-metabolic syndrome effects. These results showed that Hypericum perforatum L. has good potential to be used for the production of potential functional food with not only anti-depression properties.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/foods10061379/s1, Table S1. The parameters for all the molecules monitored by MRM method: precursor (Q1), product ions (Q2) as well as collision energy (CE) and retention times, Table S2. Antimicrobial activity of tested samples towards bacteria and yeast; MIC-minimum inhibitory concentration, MLC-minimum lethal concentrations, ns-not studied.