2.1. Reagents and Materials

The specific fluorescent probe GP-BAN was synthesized in our previous work by the author Zou et al. [28]. Vildagliptin was obtained from Shanghai Titan Technology Co Ltd (Shanghai, China). Acetonitrile was purchased from Tianjin Yongda Chemical Reagent Co Ltd (Tianjin, China). Dimethyl sulfoxide (DMSO) was purchased from McLean (Shanghai, China). Chinese medicine pellets were bought from Sanjiu Medicine. DMEM high-sugar medium was purchased from HyClone (Beijing, China). Fetal bovine serum (FBS) was purchased from Platts (Wuhan, China). Penicillin was purchased from Solarbio (Beijing, China), and HepG-2 cells were purchased from ATCC (USA).

2.2. General Procedure for DPP IV Inhibition Assays in Living Cells

Inhibition screening assays were performed by using GP-BAN which had been reported in our previous work (Figure 1) [28]. The procedure for the detection of living cells in this study is as follows. First, HepG-2 cells were cultured in DMEM high-sugar medium with 10% FBS and 1% penicillin–streptomycin solution and placed in a humidified environment (95% air and 5% carbon dioxide) at 37°C. Then, the specific fluorescent probe GP-BAN was added to each well and incubated for 60 min. The wells were then rinsed three times with PBS (pH = 7.4). The fluorescence intensity of the cells was measured using a multifunctional continuous-wave fluorometer with an excitation wavelength of 430 nm and an emission wavelength of 535 nm.

2.3. Chromatography and Analytical Conditions

The DPP IV inhibition assays in living cells were validated by the LC-UV-based chromatography method. Both GP-BAN and BAN were analyzed by a UPLC system, equipped with two independent ternary gradient UPLC pumps (integrated modules), a LPG-3400SD vacuum degasser, a WPS-3000 autosampler, a TCC-3x00 (RS) column oven, and a DAD detector. Acclaim PepMap RSLC column was used in this study, and the column temperature was maintained at room temperature. The mobile phase was a mixture of 0.1% formic acid (A) and methanol (B). The following gradient elution program was used: 0–5 min, 10% B; 5–35 min, 10%–90% B, 35–45 min, 90%–100% B, 45–55 min, and 100%–10% B. The system was operated at a flow rate of 1.0 mL/min, and the injection volume was 10 μL.

2.4. Method Validation

In the present study, the linear range for DPP IV activity detection and the precision and stability of the newly developed assay were studied in detail. First, the standard curve was constructed with nine different concentrations of probe GP-BAN (0 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 50 μM, and 100 μM) by measuring the fluorescence intensity of the cells using a multifunctional continuous-wave fluorometer, and the linearity of the method was assessed by linear regression analysis. Besides, the present method was validated by the LC-FD by the construction of a standard curve in the same conditions. The stability of the product BAN was assessed by analyzing different concentrations (5 μM, 10 μM, and 25 μM) of the BAN in the reaction mixtures for indicated time at 4°C. Incubate for 24 h using the enzyme labeling method as described in Section 2.2. The solution in the wells of the 96-well plate is then aspirated into 1.5-mL tubes. And after 48 h, the aspirated solution is reinoculated into the 96-well plate. Finally, the fluorescence intensity of the cells is measured using a multifunctional continuous-wave fluorometer with an excitation wavelength of 430 nm and an emission wavelength of 535 nm.

2.5. Statistical Analysis

All experiments were set up with three parallel samples, and all values were expressed as mean ± SD. The effect on DPP IV enzyme activity was assessed by linear relationships between variables using Prism software.

3.1. Method Development and Condition Optimization

As mentioned above, we were looking for an assay that could accurately and easily detect DPP IV enzyme activity at the live cell level. As reported in previous work, GP-BAN and BAN showed good fluorescence signal intensities at the emission wavelengths of 455 nm and 535 nm, respectively [28]. Moreover, exogenous compounds did not interfere with detection at the excitation and emission wavelengths of the probe assay. This prompted us to develop an assay based on the multifunctional microplate reader fluorescence method for live-cell detection using the fluorescent probe GP-BAN. To explore the applicability of the method and select the most suitable cell lines for DPP IV enzyme activity, the DPP IV activities of commonly used cell lines using the GP-BAN probe. As shown in Figure 2, DPP IV activity was particularly high in HepG-2 cells compared to other cell lines, which aligns with the high expression of DPP IV reported in previous studies [30]. In fact, DPP IV was overexpressed in liver injury, nonalcoholic hepatitis, and other liver-related diseases [31]. Based on these results, HepG-2 cells were selected as the most suitable live cell reaction system for the DPP IV activity assay [30].

After determining the cell line used in the present assay, the experimental conditions were subsequently systematically optimized, in terms of cell density, concentrations of probe, and incubation time. First, the concentration of the probe GP-ACN was first studied in a live cell system. HepG-2 cells were incubated with different concentrations of GP-BAN, from 0 to 200 μM. The fluorescence intensity was recorded immediately after the addition of the probe. As shown in Figure 3, the fluorescence intensity gradually increased with the increase of probe concentration during incubation with GP-BAN for 60 min. Considering the sensitivity and the cost of consumables, 50 μM was considered to be the final concentration of GP-BAN, which was superior to those previously reported in the literature [28]. Then, the reaction times of the GP-BAN probe in the HepG-2 cell system were examined. The change in fluorescence intensity with reaction time was tested by adding 50 μM GP-BAN probe to the reaction wells at 37°C. As shown in Figure 4, it is clear that the increase in incubation time resulted in a gradual enhancement of intracellular fluorescence intensity. In order to obtain higher fluorescence intensity and shorten the experiment time, 60 min was determined to be the optimal reaction time. Next, the cell density in the plate was explored. As shown in Figure 5, different cell densities were set and fluorescence detection was performed at 37°C and a probe concentration of 50 μM. Considering that cell culture should be continued after cell administration, 15,000 cells per well were determined as the cell density for subsequent experiments.

3.2. Method Verification

As mentioned above, an effort was made to construct an easy-to-use assay for the evaluation of DPP IV activity and screening of human DPP IV inhibitors in living cell system. To this end, a fluorescence microplate reader–based assay was developed utilizing a fluorescence probe of DPP IV. Then, the linear range, precision, accuracy, and stability were validated. As shown in Figures 6(a) and 6(b), the standard curves of GP-BAN and BAN exhibit a good linear relationship between concentration and fluorescence intensity within the linear concentration range of 0.2–100 μM. Meanwhile, an HPLC-based method was developed to further confirm the accuracy of the assay based on DPP IV enzyme activity at the live cell level. The GP-BAN and BAN standard calibration curves constructed based on the HPLC method are shown in Figures 6(c) and 6(d). The results show that there is a good linear relationship between the peak area and concentrations in the linear concentration range of 0.2–100 μM. The HPLC assay demonstrates a viable approach to validate the accuracy of the assay for DPP IV enzyme activity at the live cell level, which greatly reduced the interference from the endogenous matrix during fluorescence analysis. As shown in Table 1, the stability of BAN in different mixed reaction systems in samples stored at 4°C at different time points (0, 24, and 48 h) was analyzed. The results showed that BAN in the reaction system remained stable for 48 h, and the recovery rate was over 95%. Therefore, these results indicate that the method of using live cells directly as an enzyme source to detect DPP IV enzyme activity through a fluorescence microplate reader exhibits good sensitivity and stability and is suitable for the detection of DPP IV enzyme activity in complex biological samples.

3.3. Screening and Application of DPP IV Enzyme Inhibitors

Due to the excellent performance of this assay, the constructed assay based on DPP IV enzyme activity at the live cell level was utilized for rapid screening of DPP IV inhibitors. Two known DPP IV inhibitors (sitagliptin and vigabatrin) were first used to test the inhibitory effects of these two compounds on DPP IV enzyme in living cells. As shown in Figure 7, the IC₅₀ values of sitagliptin and vigabatrin in live cells were 15.97 and 0.5829 μM, respectively. And the inhibitory results were basically in line with those previously reported in the literature (IC₅₀ values of selegiline and vildagliptin in the enzyme reaction system were 0.684 μM and 2.286 μM, respectively). [32]. Chinese medicine resources are a treasure trove for finding DPP IV inhibitors, and many drug candidates are derived from natural products [33, 34]. However, the composition of Chinese medicines is complex, which makes it rather difficult to discover the active ingredients from them [35]. Accordingly, efficient screening tools are often lacking. The efficient screening system based on the cellular level constructed in this study has the potential to provide an efficient system for screening inhibitors based on DPP IV targets from traditional Chinese medicine resources. This study successfully achieved high-throughput screening of potential DPP IV inhibitors within traditional Chinese medicine. Therefore, a variety of herbal particles were first selected, and crude extracts were prepared. Subsequently, the inhibitory effects of these crude extracts on intracellular DPP IV were evaluated using the method constructed in this study. As shown in Figure 8, a variety of herbal particles showed potent inhibitory effects. In addition, some Chinese medicines showed no or weak inhibition (Figure S1). The results indicated that the method established in this study was well suited for high-throughput screening of DPP IV inhibitors in living cell.