3.1 Fluorescence spectrum of F aqueous solution
The fluorescence spectra of F aqueous solution at different pHs were measured, as shown in Fig. 1. F hardly emitted fluorescence in the range of pH 2.0–5.0. The fluorescence peak of F was significantly enhanced at the excitation wavelength (λex) of 256 nm and 334 nm and the emission wavelength (λem) of 464 nm in weak acidic and weak basic conditions (pH > 5.0). Fluorescence intensity would reach its peak and remain stable in pH range of 9.3–12.0, and then quench rapidly as the pH continued to increase (pH > 12.0), but the wavelength of excitation and emission remained the same throughout the progress.
The reason why the fluorescence spectrum changed with pH is that F molecules contain ionizable hydroxyl protons. There is a hydroxyl group at position 7 (7-OH) in the F molecule as shown in Scheme 1. The proton dissociation of 7-OH caused the change of fluorescence spectrum when the pH of the solution changed from near neutral to weakly alkaline. The ionization constant of 7-OH proton was determined to be pKa = 7.31 ± 0.03 using the pH-F data from the Fig. 1(c) and based on pH-Fluorescence method .
UV absorption spectra of F at different pH conditions were studied to verify the 7-OH proton ionization of F, and the results are shown in Fig. 2. In the pH range of 2.2–11.2, three absorption peaks appeared in the absorption spectra, which were located at 255, 303 and 334 nm, respectively. As the pH increased, the absorption peaks of 255 and 334 nm increased, while the absorption peak at 303 nm decreased, in this way, three isochromatic points located at 245, 285 and 312 nm were formed. The spectral characteristics showed that the molecular form of F changed into ionic form under weak alkaline conditions. According to pH-A data in Fig. 2 (b), the 7-OH proton ionization constant of F (pKa) was calculated by pH- spectrophotometry , the result pKa=7.34 ± 0.01 was consistent with the pKa value obtained by the fluorescence method. In addition, there were isosbestic points in Fig. 2, but no isofluorescent points in Fig. 1, which showed that both the molecule and ionic form of F can absorb light in the ultraviolet region, but only the ionic form can produce fluorescence.
As shown in Fig. 1, the fluorescence intensity of F increased in the pH regions of 6.0–10.0 and reduced rapidly when pH > 12.0. This was due to the cleavage reaction of pyrone ring in F with product of the o-hydroxyl-phenyl-benzyl ketone derivative , as exhibited in Scheme 2. This product had no fluorescence under experimental conditions.
3.2 Fluorescence spectrum of FG and its Cleavage product
Different from F, the fluorescence of FG was very weak in the acidic, neutral or weak alkaline aqueous solutions at room temperature (only weak fluorescence of FG was observed in the literature ). The absence of ionizable hydroxyl protons, and the presence of 7-position oxoside (7-OGlu) prevents FG from producing fluorescence like F, therefore, the spectral properties did not change in varying pHs .
But in the experiment, we found that FG aqueous solution would produce strong fluorescence when heated under alkaline conditions, as shown in Fig. 3. The fluorescence intensity of the FG aqueous solution was still very weak after heating in the pH range of 1.0–8.8, but when pH > 8.8, the fluorescence peak located at λex/λem = 243 / 388 nm enhanced significantly, until the fluorescence intensity reached the maximum at pH 12.5, then dropped sharply as the pH increased (pH > 12.5).
As the experimental results shown in Fig. 3, we can conclude that, chemical changes must have occurred in FG and produced new fluorescent substances under hot alkaline conditions. According to the molecular structure of FG, it can infer that there are two possible reactions: one is the ring-opening reaction of γ- pyrone ring, as shown in Scheme 2, and the other was the hydrolysis reaction of 7-OGlu.
We inferred which kind of chemical reaction happened in FG combining with the fluorescence characteristics of the reaction product. If the hydrolysis reaction of the 7-oxo-glycosidic bond had occurred, the reaction product should be like the anionic form of F after 7-OH proton ionization, as the fluorescence spectrum was shown in Fig. 1, the λex is located at 256 nm and 334 nm, and the λem is located at 464 nm. However, the λex (243 nm, 288 nm) and λem (388nm) of the actual product shown in Fig. 3 were significantly different from the wavelength position in Fig. 1. These results indicated that no hydrolysis reaction occurred under hot alkaline conditions. Another speculation, if FG had undergone the ring-opening reaction of the γ-pyrone ring, the cleavage product should has a lower degree of conjugation in the molecular structure compared to the anionic form of F, and moreover, should has shorter-wavelength fluorescence peaks according to the theory of fluorescence, the theoretical speculation was in good agreement with the results shown in Fig. 3. Therefore, it could be inferred that the ring-opening reaction of the γ-pyrone ring occurred under hot alkaline conditions, and the cleavage product is a fluorescent. But due to the hydrolysis reaction of the cleavage product, its fluorescence quenched rapidly with the increased pH when pH > 12.5, the hydrolyzate was the same as the cleavage product of the anion form of F in Fig. 2.
In summary, the fluorescence properties of F and FG were quite different, although the two are the relationship between glycoside and aglycone from the molecular structure. In the experiment, FG cannot be converted to F by the cleavage of the glycosidic bond under hot alkaline conditions, instead, the γ-pyrone ring cleavage reaction occurred first to produce fluorescence, and then the glycosidic bond hydrolysed under strong alkaline conditions, resulting in fluorescence quenching.
3.3 Effect of Solvent on Fluorescence of F
The effect of solvent (the volume fraction of methanol in aqueous solution) on the fluorescence of F was investigated under weak alkaline conditions (pH 9.3). The results in Fig. 4 show that when the volume fraction of methanol in the aqueous solution was changed, the fluorescence wavelength was almost unchanged, but the fluorescence intensity was significantly enhanced. Therefore, in this study, the volume fraction of methanol in the aqueous solution was controlled to 10% or less when studying the effect of other experimental conditions on the fluorescence.
3.4 Effect of Heating Temperature on Fluorescence of FG
As shown in Fig. 5, fluorescence of FG is extremely weak under the strong alkaline solution (pH 12.5) at room temperature, then increases along with the heating temperature increases, indicating that the temperature accelerated the cleavage reaction. When the temperature is greater than 70°C, the fluorescence reaches the maximum, showing that the cleavage reaction is basically completed. The fluorescence spectra were similar to those in Fig. 3 (a, b), and had no changes during the entire temperature-changing progress.
3.5 The stability of fluorescence
The experiments revealed that, the fluorescence of F was basically stable when placed in weakly alkaline solution at room temperature, and also had no changes when the solution was continuously irradiated by the xenon lamp.
The fluorescence intensity of FG increased with the extension of heating time and became stable after 1.5 h, and did not change significantly when continuously exposed to the xenon lamp for 200s, indicating that the fluorescence properties were basically stable.
3.6 Fluorescence Quantum Yield
Yf of F in a weak alkaline solution (pH 9.3) was measured to be 0.042, and that of FG heating pyrolysis product in alkaline solution (pH 12.5) was 0.020. Though the values of Yf were not very high, we can analysis them using modern fluorescent instruments with high sensitivity.
3.7 Relationship between concentration and fluorescence intensity
Based on the above experimental results, a series of solutions containing different amounts of F under pH = 9.27, from 0.0117 µg·mL− 1 to 1.86 µg·mL− 1, were prepared and their fluorescence spectra were scanned, as shown in inset in Fig. 6(a). A working curve of fluorescence intensity, IF (λex/λem = 334 nm / 464 nm), versus concentration of F, cF, was drawn, as shown in Fig. 6(a). In the range of 0.0117–1.86 µg·mL− 1, IF has a linear relationship with cF. The regression equation is IF = 21.9 + 2188.8c, with the correlation coefficient R2 = 0.999 (n = 14). The blank signal was scanned and the lowest limit of detection for F was found to be 2.60 ng·mL− 1 (9.69×10− 9 mol·L− 1).
Similarly, Fig. 6(b), a working curve of IF (λex/λem= 288 nm / 388 nm) versus cFG was drawn by controlling the pH of the solution to 12.5, heating in a boiling water bath for 1 hour, and scanning the fluorescence spectra (as shown in inset of Fig. 6b) after cooling to room temperature. The results show that the linear relationship between IF and cFG is good in the range of 0.0146- 2.92µg·mL− 1, the regression equation is IF = 18.9 + 611.6c, R2 = 0.998 (n = 15), and the lowest limit of detection for FG was found to be 9.30 ng·mL− 1 (2.16×10− 8 mol·L− 1).
It can be seen from the above results that when a sample contains both F and FG, although the structures of the two compounds are similar, the difference in fluorescence between the two can be used for quantitative analysis, such as controlling the sample solution to weakly alkaline conditions for the analysis and determination of F, and the sample solution can be placed under strong alkali heating conditions when determining the content of FG.