Lipase activity is significantly influenced by physiochemical factors and can exhibit distinct catalytic activities depending on specific reaction condition variants [20]. Thus, the experimental procedure for assessing the in vitro PPL catalytic activity required optimization of some experimental parameters, namely the type of buffer, enzyme, and substrate concentrations, as well as the pH value.

Notably, the analysis of the different graphs of Abs versus time presented in Fig. 4 showed that, in general, the curve exhibited linearity between 10 and 30 min of the reaction, which allows a satisfactory time-consuming experimental activity (30 min). Thus, the criteria to select the optimal conditions used to test the inhibitory activity of compounds against PPL catalytic activity included the capability of the condition to reach the Abs of 0.5, within a maximum of 30 min.

Effect of buffer composition variation on PPL catalytic activity with p-nitrophenyl butyrate (pNPB) as substrate (mean ± SEM, n ≥ 3). a Tris-buffer (100 mM Tris and 5 mM CaCl2, pH 7.4); b PBS buffer (PBS with Tween 80 (0.1%), pH 7.45)

Regarding buffer composition, Tris-buffer provides a more linear response compared to PBS with Tween (0.1%) (Fig. 4), and therefore a more easily modulated kinetics.

The next tested parameters were the enzyme and substrate concentrations. As expected, PPL catalytic activity was dependent on enzyme concentration, with the reaction rate increasing proportionally to the enzyme concentration. Notably, even at the lowest substrate concentration tested, the maximum enzyme concentration tested (2.5 mg/mL) achieved an Abs of 0.5 within 10 min. Similarly, the enzyme concentrations of 0.625 and 0.3125 mg/mL also reached the Abs of 0.5 much earlier than the established 30 min as the top maximum timepoint of linearity. Thus, the concentration of 0.156 mg/mL as the first concentration of enzyme that allowed the achievement of satisfactory enzyme activation, corresponding to an Abs of approximately 0.5, was chosen as the enzyme concentration for our study.

A parallel analysis was performed to determine and establish the optimal substrate (pNPB) concentration for the assay. Similarly to what was observed for enzyme concentrations, higher substrate concentrations resulted in increased reaction rates. Within the first 10 min, the reaction rates appeared to be relatively consistent across different substrate concentrations, except for the lower concentrations 62.5 μM (Fig. 5a) and 125 μM (Fig. 5b), where the reaction rate was lower. From 10 min onwards, differences in the reaction rate became evident, and higher absorbance values were achieved at higher substrate concentrations. Moreover, when paired with the chosen enzyme concentration, the substrate concentration of 750 μM (Fig. 5e) yielded an intermediate reaction rate, keeping the absorbance measurements within the optimal range for testing the compounds under study.

Effect of substrate p-nitrophenyl butyrate (pNPB) concentration variation on porcine pancreatic lipase (PPL) catalytic activity (mean ± SEM, n ≥ 3). a pNPB 62.5 µM; b pNPB 125 µM buffer; c pNPB 250 µM; d pNPB 500 µM; e pNPB 750 µM

Finally, as the pH level significantly influences the rate and efficiency of enzymatic reactions by affecting the enzyme's structure and its interaction with substrates, some results showed that when using the buffer at pH 7.4, the enzyme activity increased at a better rate than lower pH values within the 30 min considered initially for the reaction [20].

In this study, we also investigated the use of 4-MUO as an alternative substrate for screening PPL catalytic activity. Similarly to UV/Vis spectrophotometry, some experimental parameters were optimized (substrate and enzyme concentrations, and excitation and emission wavelengths), except for the buffer solution that suffered slight modifications according to Nakai et al. [23], and the pH value that was maintained at the value of 7.4. Moreover, as the curves also exhibited linearity at an early stage of the reaction, the experimental activity of 30 min was also maintained.

To investigate the effects of substrate concentration, the PPL-catalysed hydrolysis of 4-MUO at different initial concentrations was conducted under the same established conditions (pH 7.4 and 37 °C) for 70 min. Similarly to what was previously observed, higher substrate concentrations lead to increased reaction rates, with higher RFU values. Although all the concentrations demonstrated acceptable linearity throughout the assay, ideally the difference between the slope of the curve with and without substrate should be as significant as possible to enhance the results’ reliability. Therefore, as seen in Fig. 6, the highest substrate concentration (100 µM) showed the highest difference from the blank. However, given that the 50 µM concentration also had an acceptable difference from the blank with a lower associated error, this concentration was chosen.

Effect of substrate 4-methylumbelliferyl oleate (4-MUO) on porcine pancreatic lipase (PPL) catalytic activity (mean ± SEM, n ≥ 3). Relative fluorescence unit (RFU)

The optimization also included the enzyme concentration as one of the parameters under study. The analysis of the graphic of RFU versus time presented in Fig. 7 showed that all the tested concentrations exhibited linearity within the designated 30-min reaction time and that, as observed before, higher PPL concentrations resulted in increased reaction rates.

Effect of porcine pancreatic lipase (PPL) concentration variation with 4-methylumbelliferyl oleate (4-MUO) as substrate (mean ± SEM, n ≥ 3). Relative fluorescence unit (RFU)

The highest concentrations (0.2 and 0.4 mg/mL) exhibited the greatest difference from the blank (Fig. 7). Given that the concentration of 0.4 mg/mL showed the greatest difference, with associated errors similar to the previous concentration, this was the PPL concentration selected to proceed with the assay.

Lastly, to minimize potential interferences from the compounds under study, and to enhance signal response, wavelength values and bandwidth were optimized, using a monochromator microplate reader (Cytation 5, BIO-TEK).

Considering that preliminary assays showed that some of the compounds absorbed within the 360–380 nm wavelength range, the absorbance spectrum of the mixture (4-MUO 50 µM and PPL 0.4 mg/mL) was investigated to understand the potential use of wavelengths outside this specific range. As seen in Fig. 8, despite the slight signal reduction, wavelengths between 385 and 420 nm still appeared to be suitable for our assay.

Absorbance spectrum of porcine pancreatic lipase (PPL) with 4-methylumbelliferyl oleate (4-MUO) as substrate. Wavelength (λ)

Thus, the emission spectrum with different excitation wavelengths (from 360 to 420 nm) showed that the most favourable wavelengths ranged from 395 to 405 nm (Fig. 9a). Considering that in fluorimetric assays, an excessive amount of liberated fluorophore can generate an RFU exceeding the sensitivity of the microplate reader, wavelengths lower than 395 nm had to be excluded. On the other hand, wavelengths higher than 405 nm were also excluded since the RFU values were too low.

Effect of λex variation on porcine pancreatic lipase (PPL) with 4-methylumbelliferyl oleate (4-MUO) as substrate. a Emission spectrum; b relative fluorescence unit (RFU) versus time

To determine the best conditions for the assay, the excitation wavelengths with the highest absorption (395, 400, and 405 nm) were tested with different bandwidths. Given that the excitation wavelength should ideally be as far away as possible from the compounds’ absorption zone and that using λex = 405 ± 15 nm the signal still showed significant RFU values, this excitation wavelength was selected as the preferred condition.

Thus, from the analysis of the results presented through the emission spectrum and RFU versus time graphic (Fig. 9), the optimal conditions were defined as λex = 405 ± 15 nm and λem = 450 ± 25 nm (highlighted in Fig. 9a).

Following the assessment and determinations of optimal experimental conditions for both microanalysis systems, our study evaluated the inhibitory effects of a group of six chalcones against PPL catalytic activity, structurally defined in Table 3. Additionally, orlistat was tested as the positive control.

The chosen chalcones exhibit differences in substitution patterns at C-2’ and C-4’ of the A-ring as well as C-3, C-4, and C-5 of the B-ring. Chalcones 1–4 have different hydroxy substituents at C-2’ and C-4’ of the A-ring or C-3 and C-4 of the B-ring, while chalcone 5 and 6 have hydroxy or chloro substituents at the C-2’ or C-3 and C-4 of the A-ring and B-ring, respectively.

Our results show that, in general, the activity of the tested chalcones is dependent on the compound’s concentration and chemical structure.

As seen in Table 3, among the chalcones tested using the UV/Vis spectrophotometry microanalysis methodology, only compound 6 (a chlorinated chalcone) exhibited significant inhibitory activity with an IC50 value of 90 ± 5 µM. On the other hand, using the fluorimetric microanalysis methodology, the only compound that exhibited significant inhibitory activity was the chalcone 2 with an IC50 value of 60 ± 6 µM. On both UV/Vis spectrophotometric and fluorimetric assays, all the remaining chalcones did not show significant inhibitory activity at the maximum concentrations that could be tested without interference. Some chalcones, namely chalcone 3, (UV/Vis spectrophotometry analysis and chalcones 1 and 6 on the fluorimetric analysis) showed a very residual inhibition percentage at the maximum tested concentrations.

Finally, results presented in Table 3 show that none of the compounds that exhibited inhibitory activity against PPL demonstrate an inhibitory effect comparable to that of orlistat.