Abstract
Based on the quenching effect of emodin on the luminol-myoglobin (Mb) reaction, a sensitive method for the determination of nanogram level emodin by flow injection chemiluminescence (FI-CL) is presented for the first time. It was found that the CL intensity from luminol-Mb system could be inhibited in the presence of emodin, and the decrement of CL intensity was linearly proportional to the logarithm of emodin concentration in the range of 0.5– (R = 0.9956) with the detection limit of (3σ). At a flow rate of , a complete determination of emodin, including sampling and washing, could be accomplished in 0.5 min with the relative standard deviations (RSDs) of less than 3.5% (n = 5). The proposed method was successfully applied to the determination of emodin in pharmaceutical preparations and human serum samples. The possible CL mechanism of luminol-Mb-emodin reaction was explained.
1. Introduction
Natural ingredients that have been used traditionally to treat many diseases for hundreds of years are considered as a good choice for carcinoma therapy [1]. Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone, Figure 1, MW = 270), one of the most commonly used species, comes from dry roots of rhubarb which is a group of plants that belongs to the genus rheum in the polygonaceae family. Emodin has been extensively researched for its traditional pharmacological effects, mainly in the treatment of constipation and menoxenia, skin burn, gallstone, hepatitis, inflammation, and osteomyelitis [2, 3]. Recently, a number of studies have placed emodin back into the attention focused on its anticancer activities against several types of cancer cells and virus, for instance, inhibiting ATP-induced macrophage death [4], mediating cytotoxicity in human nonsmall cell lung cancer cells [5], inducing resensitization of lung cancer cells to anoikis [6], against β-amyloid-induced neurotoxicity, herpes simplex virus, human chronic myelocytic leukemia K562 cell lines [7–9], and inhibiting the growth of hepatoma cells [10].
The different methods for the determination of emodin have been reported, including high-performance liquid chromatography (HPLC) [11, 12], capillary electrophoresis (CE) [13], ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) [14], electrochemistry (EC) [15], UV-Vis spectrophotometry [16], and electrochemiluminescence (ECL) [17, 18]. However, there was no report on the determination of emodin using flow injection chemiluminescence (FI-CL) up to now.
It has been reported that Mb, which contains a single iron protoporphyrin or heme moiety in the ferric state Mb (FeIII), can react with luminol yielding CL emission [19, 20]. In this paper, it was found that the CL intensity from luminol-Mb system could be inhibited in the presence of emodin, and the decrement of CL intensity was linearly proportional to the logarithm of emodin concentration in the range of 0.5–300 ng·mL−1 ( = 0.9956) with the relative standard deviations (RSDs) of less than 3.5% (). The simple and rapid method was successfully applied to the determination of emodin in pharmaceutical preparations and human serum samples.
2. Experimental Section
2.1. Apparatus and Reagents
The experiment instrument was flow injection CL analyzer (model IFFL-DD, Xi’an Remax Electronic Science-Tech. Co. Ltd); PTFE tubing (1.0 mm i.d.) was used as connection material in the flow system; luminol was used as supplied to prepare a 2.5 × 10−2 mol·L−1 stock standard solution by dissolving 0.44 g luminol (Fluka, Biochemika) in 0.1 mol·L−1 sodium hydroxide in a 100 mL calibrated flask; horse heart Mb (Sigma) was purchased from local market and used as received without further purification. All reagents were of analytical purity grade. Doubly distilled water purified in a Milli-Q system (Millipore, Bedford, MA) was used throughout.
2.2. Procedures
The flow injection system used in this work was shown in Figure 2. A peristaltic pump was used to pump each of all flow streams at a flow rate of 2.0 mL·min−1. The carrier used in flow injection system was water. Luminol was used as supplied to prepare a 2.5 × 10−5 mol·L−1 solution, and NaOH solution was 2.5 × 10−2 mol·L−1. A six-way valve with loop of 100 μL was used for luminol after a stable baseline was recorded, the CL signal produced in the CL emission cell when mixed with Mb. The mixed solution was delivered into the CL cell, producing CL emission, and detected by the photomultiplier tube (PMT). The concentration of emodin could be quantified on the basis of the decrement of CL intensity, , where and were CL signals in the presence and in the absence of emodin, respectively.
2.3. Sample Preparation
Five tablets of SANHUANG PIAN (produced by Shanxi Yabao Pharmaceutical Group Industry. Co. Ltd.) were weighed and ground to fine powder using a pestle and mortar and then homogenized; a sample equivalent to approximately 425 ± 10 mg of emodin was weighed accurately and dissolved in some ethanol and then diluted with doubly deionized water in a calibrated flask and ultrasound for 10 minutes. The solution was filtered by an ordinary filter paper, and suitable filtrate aliquots from this solution were diluted to an emodin concentration within the calibration range.
3. Results and Discussion
3.1. The Relative CL Intensity-Time Profile
The behavior of the CL reaction, carried out in the absence and presence of emodin, was tested using 5.0 × 10−8 mol·L−1 Mb and 2.5 × 10−5 mol·L−1 luminol in 2.5 × 10−2 mol·L−1 NaOH solution. It can be seen from Figure 3 that CL intensity reaches the maximum at 8 s after sampling and vanishes within 25 s; the CL evidently decreases with the increasing concentration of emodin.
3.2. Effect of Luminol, Mb, and NaOH Concentration
Under different concentrations of luminol from 1.0 × 10−6 to 1.0 × 10−4 mol·L−1 and Mb solutions from 1.0 × 10−8 to 1.0 × 10−6 mol·L−1, the CL intensity was examined. It was found that the CL intensity arrived at the maximum with 2.5 × 10−5 mol·L−1 luminol and 5.0 × 10−8 mol·L−1 Mb. Thus the luminol solution of 2.5 × 10−5 mol·L−1 and Mb solution of 5.0 × 10−8 mol·L−1 were used in subsequent experiments.
Owing to the nature of the luminol reaction, which is more favorable under alkaline conditions, NaOH was introduced into the luminol solution to improve the sensitivity of the system. A series of NaOH solutions with different concentrations from 0.01 to 0.1 mol·L−1 were tested, respectively. The plot of CL intensity versus NaOH concentration reached the peak at 2.5 × 10−2 mol·L−1, and this concentration was employed in the subsequent experiments.
3.3. Effect of Flow Rate and the Length of Mixing Tubing
It was found that the CL intensity increased with the increasing flow rate. As a compromise between good precision and lower reagent consumption, 2.0 mL·min−1 of flow rate on each line was recommended. It was found that 10 cm mixing tube afforded the best results with the good sensitivity and reproducibility. Accordingly, 10 cm was selected as the optimum length of mixing tubing.
3.4. Performance of Presented Method for Emodin Determination
Under the optimum conditions described, the calibration graph of CL intensity was linear with emodin concentration in the range of 0.5–300 ng·mL−1, given the regression equation , with detection limit of 0.2 ng·mL−1 (3σ) and RSDs of less than 3.5%.
3.5. Interference Study
Under optimum conditions, the effect of foreign substances was investigated by analyzing a standard solution of emodin (50 ng·mL−1) to which increasing amounts of interfering substances were added. The tolerable limit of a foreign species was taken as a relative error of less than 5.0%. The excipients commonly found in the pharmaceutical tablets, such as starch, lactose, cellulose, stearic acid, agar, talc, fructose, and sucrose did not interfere with the determination at tolerable concentrations of 10 μg·mL−1, and the tolerable concentrations for other analytes were 5.0 μg·mL−1 for , , I-, , 2.5 μg·mL−1 for , , , ethanol, glutin, 0.1 μg·mL−1 for Cu2+, Zn2+, Cr3+ and Fe2+/Fe3+, respectively.
3.6. Possible Luminol-Mb-Emodin CL Mechanism
The possible CL mechanism of luminol-Mb-emodin reaction was discussed by fluorescence and CL. It can be seen that a strong fluorescence emission of Mb ( nm) was quenched evidently in the presence of emodin as shown in Figure 4. The linear equation was , where and were the fluorescence intensity of 1.0 μmol·L−1 Mb in the absence and in the presence of emodin with its concentration ranging from 0.1 to 10 μmol·L−1. From Stern-Volmer equation [21], the quenching rate constant L·mol−1·s−1 was obtained which was far greater than the maximum scatter collision-quenching constant of various quenchers with biopolymers 2.0 × 1010 L·mol−1·s−1 [22]. It was deduced that the fluorescence quenching of Mb with emodin was initiated by forming the ground-state complex and resulted in the CL intensity of luminol-Mb system decreased as presented in Figure 3.
4. Application
4.1. Determination of Emodin in Pharmaceutical Preparations
Following the procedure detailed above, the proposed method was applied in the determination of emodin in pharmaceutical preparations SANHUANG Tablets. The results were listed in Table 1 showing that the recovery rate ranged from 93.3 to 105.0% with the RSDs of less than 3.0% ().
4.2. Determination of Emodin in Spiked Human Serum Samples
Emodin in the spiked human serum samples prepared in the experimental section was measured, and the results were summarized in Table 2. It can be seen that the recoveries for the determination of emodin were from 94.0 to 106.3% and the RSDs were less than 3.5%, confirming the applicability of this method for emodin determination in biological samples.
5. Conclusion
A simple and sensitive CL procedure was established based on the quenching effect of emodin on the luminol-Mb CL system for the determination of emodin in pharmaceutical preparations. A comparison of the proposed CL procedure with other methods was presented in Table 3. It is clear that the proposed CL method offers the highest sensitivity and the least reagent consumption for the determination of emodin in pharmaceutical preparations and biofluids.
Acknowledgments
The authors gratefully acknowledge the financial support from Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, China, Shaanxi Province Nature Science Foundation (no. 2006B05), the NWU Graduate Innovation and Creativity Funds (no. 10YZZ29 and 09YZZ45), and the NWU Graduate Experimental Research Funds, China (no. 09YSY18).