You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


1. Introduction
===============

The family Asteraceae is the largest Angiosperm group, consisting of approximately 23,000 species distributed in 1,535 genera. It has a cosmopolitan distribution, and found on all the continents except Antarctica. South America is home to about 20% of the existing genera. In Brazil, there are approximately 180 genera and 3,000 species distributed throughout the country.

Plants from this family have been extensively studied for their chemical composition and biological activity and some have led to the development of new drugs and insecticides \[[@B1-molecules-16-04828],[@B2-molecules-16-04828],[@B3-molecules-16-04828],[@B4-molecules-16-04828],[@B5-molecules-16-04828],[@B6-molecules-16-04828],[@B7-molecules-16-04828],[@B8-molecules-16-04828],[@B9-molecules-16-04828],[@B10-molecules-16-04828],[@B11-molecules-16-04828],[@B12-molecules-16-04828],[@B13-molecules-16-04828]\].

*Praxelis clematidea* R.M. King & Robinson belongs to the Eupatorieae tribe of the family Asteraceae, and consists of 2,400 species distributed in 170 genera \[[@B14-molecules-16-04828]\]. The species has the following synonyms: *Eupatorium clematideum* Griseb. and *Eupatorium urtifolium* var. *clematideum* (Griseb.) Hieron ex. Kuntze.

It is a perennial weed native to South America and distributed throughout Bolivia, Peru and Argentina. In Brazil, it is found mainly in the states of Bahia, Alagoas, Pernambuco, Paraiba, Amazonas and Mato Grosso \[[@B15-molecules-16-04828]\]. In phytochemical studies, Bolhmann and coworkers \[[@B16-molecules-16-04828]\] isolated *N*-(acetoxy)-jasmonoylphenylalanine-methyl-ester. Gas chromatographic analysis showed the presence of sesquiterpenes and monoterpenes in the essential oil extracted from *P. clematidae*, which also showed growth inhibitory effect on two plant species, *Lactuca sativa* and *Brassica campestris*, and on fungal colonies of *Fusarium oxysporum* and *Phytopthora capsici* \[[@B17-molecules-16-04828]\]. A pharmacological study conducted with the aerial parts of this species demonstrated significant gastroprotective activity against gastric ulcers induced in animals with ethanol, stress, and a non-steroidal antiinflammatory \[[@B18-molecules-16-04828]\].

The studies on *Praxelis clematidea* don't report the presence of flavonoids, although scientific studies conducted on the family Asteraceae have identified flavonoids as important chemotaxonomic markers of this family \[[@B19-molecules-16-04828]\]. Based on this information, we started with the aerial parts of *Praxelis clematidea* to isolate compounds belonging to this secondary metabolite class. This class is increasingly becoming an object of investigation, and many studies have isolated and identified flavonoids that possess antifungal, antiviral and antibacterial activities. In addition, various studies have demonstrated synergy between active flavonoids, and between flavonoids and conventional chemotherapeutic agents \[[@B20-molecules-16-04828],[@B21-molecules-16-04828]\].

The ever increasing bacterial resistance to antibiotics is a serious problem for public health that affects most current antibacterial agents. Efflux pumps are integral proteins of the bacterial membrane and are recognized as one of the major sources of bacterial resistance since they extrude antibiotics from the cell \[[@B22-molecules-16-04828],[@B23-molecules-16-04828]\].

Modulators of antibiotic drug resistance are compounds that potentiate antibiotic activity against resistant strains. Some of these agents act as efflux pump inhibitors (EPIs) \[[@B24-molecules-16-04828],[@B25-molecules-16-04828]\]. Plants provide a rich source of EPIs and several compounds have been identified as potent inhibitors \[[@B26-molecules-16-04828],[@B27-molecules-16-04828],[@B28-molecules-16-04828]\].

The aim of the present work was to isolate and characterize the structure of flavonoids from *Praxelis clematidea* and study their activity as modulators of drug resistance in *Staphylococcus aureus* SA-1199B.

Some methoxylated flavonoids that potentiate the activity of antimicrobial drugs have already been described \[[@B29-molecules-16-04828],[@B30-molecules-16-04828],[@B31-molecules-16-04828],[@B32-molecules-16-04828]\]. However, as far as we know, none of the flavonoids presented here has been previously evaluated. The results add new scientific evidence that flavonoids modulate antibiotic resistance, probably by efflux pump inhibition.

2. Results and Discussion
=========================

The structural identification of the compounds ([Figure 1](#molecules-16-04828-f001){ref-type="fig"}) was carried out based on the analysis of the spectral data and by comparison with the literature \[[@B33-molecules-16-04828],[@B34-molecules-16-04828]\]. The compounds were: (1) apigenine (4',5,7-trihydroxyflavone), (2) genkwanin (4',5-dihydroxy-7-methoxyflavone), (3) 7,4'-dimethylapigenin (5-hydroxy-4',7-dimethoxyflavone), (4) trimethylapigenin (4',5,7-trimethoxyflavone), (5) cirsimaritin (4',5,-dihydroxy-6,7-dimethoxyflavone) and (6) tetramethylscutellarein (4',5,6,7-tetramethoxyflavone).

![Isolated flavonoids from *Praxelis clematidea*.](molecules-16-04828-g001){#molecules-16-04828-f001}

Methoxylated flavones showed no antibacterial activity at 256 μg/mL against the tested strain of *S. aureus*. When the compounds were added to the growth medium at 64 µg/mL (1/4 MIC), a reduction in the MIC of at least two-fold (and up to 16-fold) was observed for norfloxacin and ethidium bromide ([Table 1](#molecules-16-04828-t001){ref-type="table"}). All experiments were carried out at least twice with consistent results.

molecules-16-04828-t001_Table 1

###### 

Minimum inhibitory concentrations (MICs) of antibiotics and ethidium bromide against *Staphylococcus aureus* strain SA-1199B, in the absence and presence of flavones.

  Flavones   MIC (µg/mL)             
  ---------- ------------- --------- ----
  None       128           32        16
  **1**      128           32        16
  **2**      64 (2×) ^a^   16 (2×)   16
  **3**      64 (2×)       16 (2×)   16
  **4**      16 (8×)       8 (4×)    16
  **5**      32 (4×)       8 (4×)    16
  **6**      8 (16×)       2 (16×)   16

^a^ Fold reduction in MIC.

Methoxylated flavones modulate drug activity by reducing the concentration needed to inhibit the growth of the drug-resistant (effluxing) bacteria. This activity may be related to flavanoid lipophilicity due to the presence of methoxyl groups. Lipophilicity is a common feature of several efflux pump inhibitors and may be a key factor for inhibition in Gram-positive bacteria \[[@B28-molecules-16-04828]\].

Ethidium bromide is a well-known substrate for the NorA efflux protein, and active efflux is the only known mechanism of resistance to this DNA-intercalating dye \[[@B35-molecules-16-04828]\]. Therefore, the use of ethidium bromide against the strain SA-1199B was used to demonstrate that the methoxylated flavones evaluated here modulated norfloxacin resistance by efflux pump inhibition.

Pefloxacin, a hydrophobic quinolone, is a poor substrate of the NorA efflux pump, and it was used as a negative control \[[@B25-molecules-16-04828]\]. Reductions in MICs of norfloxacin and ethidium bromide when combined with chlorpromazine or trifluoperazine were also observed (data not shown), and the results were consistent with those reported by Kaatz *et al.* \[[@B24-molecules-16-04828]\] and by Falcão-Silva *et al.* \[[@B32-molecules-16-04828]\]; both phenothiazines were used as positive (internal) control.

The results can be explained by the increasing lipophilicity in the compounds. An analysis of log P values for the compounds (calculated with ChemDraw Ultra 10.0, Cambridge Software) revealed the following order of lipophilicity: **1** (log P 1.9) \< **5** (log P 2.04) \< **2** (log P 2.17) \< **3** (log P 2.43) \< **6** (log P 2.57) \< **4** (log P 2.69). This order explains, in part, the following order of activity: Nor: **1** \< **2**/**3** \< **5** \< **4** \< **6** and EB: **1** \< **2**/**3** \< **5**/**4** \<**6**.

The importance of the presence of a methoxyl in the 4' position \[[@B29-molecules-16-04828]\] was also observed, as the activity of compounds **3** and **4** was higher than that of compounds **1** and **2**, as well as the activity of compound **6** in relation to that of compound **5**. Another important factor was the total number of methoxyls in the flavonoids, in general, compounds **5** and **6** were more active than the other compounds, which is evident when we observe that compound **5** has a greater lipophilicity only compared to compound **1** and does not show a methoxyl in the 4' position, even though it is more active than compounds **1**, **2** and **3**.

3. Experimental
===============

3.1. General
------------

The NMR spectra were obtained with a Mercury-Varian spectrometer at 200 MHz (^1^H) and 50 MHz (^13^C) and a VARIAN System model operating at 500 MHz (^1^H) and 125 MHz (^13^C). The solvents used were CDCl~3~, CD~3~OD and DMSO-*d~6~*, whose characteristic peaks in ^1^H and ^13^C-NMR are used to adjust the frequency scale. For column chromatography, silica gel 60 (70-230 Mesh) from Merck was utilized as the stationary phase. PF~254~ silica gel from Merck was used for the analytical (ATLC) and preparative (PTLC) thin-layer chromatography. The studied substances were identified by using ultraviolet radiation at wavelengths of 254 and 366 nm and by impregnation of plates in glass containers saturated with iodine vapor.

3.2. Plant Material
-------------------

The aerial parts of *Praxelis clematidea* R.M. King & Robinson were collected in Lagoa do Paturi, a municipality of Santa Rita, in the state of Paraiba (Brazil), in May 2008. The identification of the botanical material was performed by Prof. Dr. Maria de Fatima Agra, Botany Sector, Laboratory of Pharmaceutical Technology/UFPB "Professor Delby Fernandes de Medeiros". Exsiccates of the plant are deposited in the Prof. Lauro Pires Xavier (JPB) Herbarium, Paraiba Federal University, under the code M. F. Agra *et al*. 6894 (JPB).

3.3. Extraction and Isolation
-----------------------------

The dried and pulverized plant material (aerial parts, 10 kg) was submitted to exhaustive maceration utilizing ethanol as the extraction solvent (3 × 10 L, every 72 h). The ethanolic solution obtained was concentrated in a rotary evaporator under reduced pressure, resulting in a crude ethanolic extract (600 g). This was partitioned with hexane, chloroform and ethyl acetate. The chloroform phase (120.96 g) was submitted to adsorption column chromatography (CC) using silica gel as the stationary phase and chloroform and methanol as the mobile phase, both as pure or as binary mixtures of increasing polarity, resulting in 188 fractions. These samples were analyzed by ATLC, and after examination with under UV light and iodine vapor, they were classified according to their Rf values into 25 groups. The sub-fractions 10--18 and 64--70 appeared as yellow solids and were identified as compounds **3** and **5**. The subgroup 23--24 was submitted to PTLC, utilizing chloroform and methanol (97:3) and furnishing compounds **6**, **2** and **4**. The subgroup 77--80 was submitted to PTLC, using chloroform and methanol (95:5) and supplying compound **1**.

3.4. Bacterial Strains
----------------------

The *S. aureus* strain used, SA-1199B, over expresses the norA gene encoding the NorA efflux protein which extrudes hydrophilic fluoroquinolones and other drugs such as DNA-intercalating dyes \[[@B24-molecules-16-04828],[@B36-molecules-16-04828]\]. The strain, kindly provided by Professor Simon Gibbons (University of London), was maintained on blood agar base slants (Laboratorios Difco Ltda., Brazil), and prior to use, the cells were grown overnight at 37 °C in brain heart infusion broth (BHI--Laboratorios Difco Ltda., Brazil).

3.5. Antibiotics and Chemicals
------------------------------

Norfloxacin, pefloxacin and ethidium bromide were obtained from Sigma Aldrich Co. Ltd. (USA). The stock solutions of the flavones were prepared in DMSO, and the highest concentration remaining after dilution in broth (4%) caused no inhibition of bacterial growth.

3.6. Drug Susceptibility Testing and Modulation Assay
-----------------------------------------------------

The minimum inhibitory concentrations (MICs) of the antibiotics and flavonoids were determined in BHI by micro-dilution assay, using a suspension of ca. 105 CFU/mL and a drug concentration range from 256 to 0.5 μg/mL (twofold serial dilutions). MIC is defined as the lowest concentration at which no growth is observed. A solution of resazurin (0.01% w/v in sterile distilled water) was used to detect bacterial growth by color change from blue to pink. For the evaluation of flavones as modulators of drug resistance, the "modulation assay" was used, a method that has been widely applied to identify potential EPIs \[[@B29-molecules-16-04828]\], *i.e.*, the MICs of antibiotics were determined in the presence of the flavones (in the BHI) at a sub-inhibitory concentration.

3.7. Log P Estimation
---------------------

The structures were drawn utilizing the ChemDraw Ultra® 10.0 program (CambridgeSoft, 1986--2005), which also estimate their log P values.

4. Conclusions
==============

Six flavones were isolated from *Praxelis clematidea* and identified through ^1^H and ^13^C-NMR data. Assays were carried out with these compounds against a strain of *Staphylococcus aureus* SA-1199B, with NorA efflux pump activity, demonstrating that the highest methoxylated flavones showed the highest efflux pump inhibition, or modulating of bacterial resistance. Inhibition of the bacterial transporter is related to the lipophilicity of the compound and might confer selectivity when used with antimicrobials.

J.P.S-J. and V.S.F-S. are very grateful to Simon Gibbons (University of London). The authors thank Maria de Fatima Agra for botanical identification of the species, and CNPq, CAPES, PRONEX/FAPESQ-PB Brazil for financial support.

*Sample Availability:* Samples of the compounds are available from the authors.