Fluorescent Properties of Hymecromone and Fluorimetric Analysis of Hymecromone in Compound Dantong Capsule

Fluorescence spectra of hymecromone (4MU) aqueous solutions are investigated at different pHs. Two fluorescent species of 4MU, neutral molecular form and anion form, are considered to be the main fluorescent forms. Quantum yields of the two forms are measured to be 0.74 at pH 5.98 and 0.95 at pH 9.75, respectively.The ionization constant of 7-hydroxyl proton of 4MU is determined to be pK a = 7.85 ± 0.03 by a pH-fluorescence method. Addition of methanol into 4MU aqueous solution leads to a blue shift of maximum emission wavelength from 445 nm to 380 nm, and a decrease in fluorescence intensity. 3D fluorescence spectra of Chinese patent drug Compound Dantong Capsule (CDC) and its four component herbal drugs are also investigated. Based on their fluorescent properties, a novel fluorimetric method is proposed for the selective determination of 4MU in CDC without preseparation. The new method is suitable for the routine quality evaluation of CDC.

The chemical composition of CDC includes 4MU (also named Dantong) and four kinds of Chinese herbal medicines, including Herba Isodonis Lophanthoidis (Xihuangcao), Herba Artemisiae Scopariae (Yinchen), Herba Andrographis (Chuanxinlian), and Rhei Radix et Rhizoma (Dahuang). The content of 4MU is one of the main quality indexes of CDC [21]. Earlier, the spectrophotometric method was applied for the determination of 4MU in CDC according to the absorbance of 4MU at 372 nm [19]. Later, since the interference of coexistent components, high performance liquid chromatographic methods (HPLC) were developed for the determination of 4MU in CDC [21][22][23]. So far, no fluorimetric method was reported for the determination of 4MU in CDC and other medicinal samples.
Fluorimetry has been recognized as one of the most useful analytical methods owing to its high sensitivity, good selectivity, simplicity, speediness, and low cost. It can be suitable for analysis of some complex samples such as traditional Chinese medicine containing a fluorescent component, especially in the case of a routine analysis. Three-dimensional fluorescence spectra (3D fluorescent fingerprint) have been used in our lab for qualitative identification of Chinese herbal medicines [24]. Moreover, a number of fluorimetric methods have been developed for quantitative determination of active components in medicinal materials, such as paeonol in Cynanchi Paniculati Radix (Xuchangqing) [25], camptothecin in common Camptotheca fruit (Xishuguo) [26], and arctiin in Arctii Fructus (Niubangzi) [27]. In this study, the fluorescent properties of 4MU are investigated, and its quantum yield and 7-hydroxyl proton ionization constant are measured. The fluorescent properties of CDC and its four consisting Chinese herbal medicines are also studied. Based on the spectral differences between 4MU and other components in CDC, a fluorimetric method is proposed for the determination of 4MU in CDC sample without preseparation.

Experimental
2.1. Apparatus. Fluorescence measurements were performed on a Hitachi (Tokyo, Japan) F-7000 spectrofluorimeter equipped with a xenon lamp, 1 cm quartz cell, and a UV-29 filter placed into the emission light path to remove secondary spectrum. The excitation and emission slits (band pass) 5 nm/5 nm were used throughout the work. Absorption spectra were recorded using a Shimadzu (Kyoto, Japan) UV-2501PC recording spectrophotometer with 1 cm quartz cell. An Orion (Beverly, USA) 868 pH/ISE meter was used for pH measurement. and diluted to appropriate concentration with 0.05 mol L −1 H 2 SO 4 when it was used. Britton-Robinson buffer solution was a mixture of phosphoric acid, boric acid, and acetic acid (each 0.02 mol L −1 ) and adjusted to an appropriate pH by addition of 0.1 mol L −1 NaOH solution. All the buffer chemicals were of analytical grade. The water used throughout the study was doubly-deionized and verified to be free from fluorescence.

General Procedure for Spectral Measurement.
A series of 10 mL volumetric flasks was added appropriate amount of 4MU or CDC and buffer solutions. The mixtures were diluted to the mark with water and mixed well. Fluorescence or absorption spectra were measured at room temperature. Meanwhile, Raman scattering of water at excitation wavelength of 350 nm was measured (expressed as R, for calibration of 3D fluorescence spectra) [24].

Determination of Ionization Constant.
A number of solutions containing the same concentration of weak acid HB and different pH were prepared. Fluorescence intensity and pH of each solution were measured. Then the ionization constant p was calculated according to the following equation [28]:  where HB and B were the fluorescence intensity of HB and its conjugate base B; they can be measured at sufficient acidic pH where all HB species exist in the neutral molecular form and at sufficient alkaline pH where all HB species exist in the anion form, respectively.

Measurement of Fluorescence Quantum Yield.
Quinine bisulphate was used as a reference (quantum yield 0.55 at excitation wavelength of 313 nm) in measuring quantum yield of 4MU. For the measurement, quinine bisulphate and 4MU solutions were prepared in proper concentration so that the absorbance ( ) of the two solutions was similar and not larger than 0.05. Absorption and fluorescence spectra were recorded, and then quantum yields were calculated according to the following equation [28,29]: where and were the fluorescence quantum yield of unknown and the reference, and were the integral fluorescence intensity of unknown and reference solutions, and and were the absorbance of unknown and reference solutions at their excitation wavelengths, respectively.
2.6. Sample Preparation. Dissolve 9.21 mg CDC powder in 100 mL methanol and dilute to 0.921 g mL −1 with methanol as sample solution.

Fluorescence Properties of 4MU
3.1.1. 3D Fluorescence Spectra of 4MU. 3D (three-dimensional) fluorescence spectra of solvent blank and 4MU aqueous solutions at pH 5.98 and at pH 9.75 were measured as shown in Figure 1. The spectra indicated that the solvent used in this study was basically no fluorescence. 4MU can produce strong fluorescence in near neutral condition with maximum excitation wavelength ( ex ) of 320 nm and maximum emission wavelength ( em ) of 445 nm, while in weak alkaline condition, the fluorescence intensity enhanced, ex red shifted from 320 nm to 360 nm, but em remained constant. Figure 1 revealed that pH has an important influence on fluorescence.

Effect of pH on Fluorescence.
As shown in Figure 2, effect of pH on fluorescence excitation and emission spectra of 4MU at various pHs were studied. In acidic conditions (pH 1.97-6.72), along with the decrease in pH, fluorescence intensity declined, ex centered at 320 nm, and em red shifted slightly from 445 nm to 455 nm. In the range of pH 7.12-10.43, along with the increase in pH, fluorescence emission at 445 nm enhanced, but the excitation band centered at 320 nm declined, meanwhile a new excitation band centered at 360 nm emerged and an iso-fluorescence point [30] formed at 330 nm. In the range of pH 10.80-13.38, along with the increase in pH, fluorescence emission at 445 nm gradually quenched while ex and em remained constant. The relationship between pH and fluorescence intensity was summarized in Figure 2(d).
The variation of fluorescence spectra implies that the solution equilibrium or molecular structure of 4MU changed along with the change in pH. According to Figure 2, we suppose that the proton dissociation and hydrolysis process of 4MU is as shown in Figure 3.
The structure of 4MU contains a benzene ring (linked with 7-OH) and a lactonic ring (include lactone bond and 2-C=O). In strong acid conditions, the 2-carbonyl oxygen protonated [28,30] to form a cationic species (type I), leading to a decrease in fluorescence intensity and a red shift in emission wavelength. In near neutral conditions (pH 4.0-7.0), 4MU exist mainly as molecular form (type II) which have strong fluorescence with ex of 320 nm and em of 445 nm. In weak alkaline conditions (pH 9.0-12.0), 7-hydroxyl proton dissociated and 4MU exist mainly as anion form (type III) which have stronger fluorescence with ex of 360 nm and em of 445 nm. In strong alkaline conditions (pH > 12.0), the hydrolysis of lactone bond take place [28,31], leading to a fluorescence quenching.

Ionization Constant of 7-Hydroxyl Proton.
With the above discussion, ionization constant of 7-OH proton can be determined in suitable conditions. Using the pH-fluorescence method described in Section 2.4, the ionization constant of 7-hydroxyl proton was determined to be p = 7.85 ± 0.03 (Table 1).  In addition, according to the relationship between fluorescence intensity and pH, we can graphically estimate the ionization constant. In Figure 2(d), the abscissa of the intersection of curve (a) and curve (b) is about 7.85, which is just the p .
The ionization constant determined above is close to the ionization constant of umbelliferone; p = 7.61 ± 0.03 [28]. The little difference between the two constants reflects the impact of 4-methyl on the acidity of 7-hydroxyl proton. declined, while a new emission band centered at 380 nm emerged. When the percentage of methanol reached 100%, the em blue shifted to 380 nm; however, the fluorescence intensity was less than that in aqueous solutions. Therefore, we choose water as the appropriate solvent in establishing a fluorimetric method in the following study, and control the methanol percentage in aqueous solution no more than 20% to obtain sensitive and stable fluorescence.

Measurement of Fluorescence Quantum Yield of 4MU.
Using the method described in Section 2.5, quantum yields of 4MU in near neutral (pH 5.98) and weak alkaline (pH 9.75) conditions were measured, as shown in Table 2. In near neutral solution, 4MU exist as molecular form; its quantum yield at excitation wavelength 320 nm was measured to be 0.74. In weak alkaline solution, 4MU exist as anion form; its quantum yield at excitation wavelength 360 nm was measured to be 0.95. These results indicate that 4MU is an excellent fluorophore. A fluorimetric method for determination of 4MU in Chinese patent drug should be very sensitive either in near neutral or in weak alkaline conditions.

3D Fluorescence Spectra of Four Kinds of Chinese Herbal Medicines in CDC.
The key point for developing a fluorimetric method is to understand the fluorescent properties of the analyte and other components in the sample, then find a way to avoid interference of the coexistent components on the fluorescence of the analyte. For these reason, 3D fluorescence spectra of four kinds of Chinese herbal materials in CDC were measured and shown in Figure 5. From Figure 5 we showed that all the four kinds of Chinese herbal medicines contain fluorescent components. The interference of these components on the fluorescence of 4MU seems to be a serious problem. However, we noted  that the concentrations of these herbal medicines in Figure 5 (400 g mL −1 ∼ 100 g mL −1 ) were much higher than the concentration of 4MU in Figure 1 (18.5 ng mL −1 ). So, whether these herbal medicines in CDC interfere with the fluorescence of 4MU should be considered further from the view point of concentration. Figure 6 shows the 3D fluorescence spectra of CDC dilute solutions at pH 5.85 and at pH 9.22. Comparing Figure 6 to Figure 1, we figured out that the two pictures are basically the same. The fluorescence peaks presented in Figure 5 are not emerged in Figure 6. This result indicated that the fluorescence of those four herbal medicines in CDC is too weak to be seen, either because of their low concentration or due to their poor luminous ability. So, we can conclude that the coexistent components in CDC do not interfere with the fluorescence of 4MU. The content of 4MU in CDC can be determined simply by a fluorimetric method. From Figures 6 and 2, we also know that the fluorimetric method may be performed at near neutral or at weak alkaline pH. For simplicity, we prefer the method to be performed at near neutral pH so that the fluorescence intensity of CDC water solution can be measured directly without using a buffer solution.

Determination of 4MU in CDC by a Standard Curve
Method. A series of standard aqueous solutions containing different amounts of 4MU, from 1.55 to 31.0 ng mL −1 , were prepared. Fluorescence spectra, as shown in Figure 7(a), and fluorescence intensity at measuring wavelength of ex / em = 320 nm/445 nm of each solution were measured. A linear calibration curve of fluorescence intensity against concentration was plotted, as shown in Figure 7(b). The regression equation obtained was = 5.5 + 104.1 (ng mL −1 ), with correlation coefficient = 0.9998 ( = 13).
A number of solutions containing different amounts of CDC were prepared and their fluorescence intensities at wavelength of ex / em = 320 nm/445 nm were recorded. Using the calibration curve, the 4MU content in CDC sample was determined to be 33.35 ± 0.10%, as shown in Table 3.

Determination of 4MU in CDC by a Standard Addition
Method. As shown in Figure 8, a standard addition method was employed to verify the result obtained above. The regression equation obtained was = 636.9 + 105.3 (ng mL −1 ), with correlation coefficient R = 0.9998 ( = 5). The content of 4MU in CDC sample was calculated to be 33.3%, which was coincident with the result obtained by standard curve method.  According to the CDC drug label, the content of 4MU in CDC is 100 mg per grain (0.3 g). It is evident that the analytical result of this method agrees with the drug label.

Conclusion
Hymecromone (4MU) is an excellent fluorophore. Acidity and solvent have important influence on its fluorescence. In near neutral aqueous solutions, 4MU exist mainly as neutral molecular form which can be produced strong fluorescence at 445 nm. In weak alkaline solutions, 4MU exist mainly as anion form which can be produced stronger fluorescence at 445 nm. In methanol solution, the fluorescence peak at 445 nm blue shifted to 380 nm and the fluorescence intensity declined in a certain extent. There is a good linear relationship between fluorescent intensity and 4MU concentration. The 3D fluorescence spectra of Chinese patent drug CDC dilute solutions are very similar to the 3D fluorescence spectra of 4MU, indicating that the main fluorescent component in

Conflict of Interests
The authors declare that they have no conflict of interests.