Chu Haihong, Di Junwei, Xu Yang, Hu Yanwei^#, Tu Yifeng
(College of Chemistry and Chemical Engineering; ^#Pharmaceutical Department of Medicine College, Suzhou University, Suzhou 215006, China)

Support by the National Natural Science Foundation (No. 20275025) would be gratefully acknowledged.

Abstract Based on the works of the enhanced electrochemiluminescence (ECL) behavior of luminol by iodide in neutral medium, the quenching effect from the flavonoids due to their reductibility for the free radicals of this enhanced ECL system was studied. It provided an excellent basis to detect the flavonoids. The ECL intensity negatively responded to the concentrations of flavonoids in the phosphate buffer solution (pH 6.7) which containing 2.0×10^-7mol/L of luminol and 2.0×10^-7mol/L of iodide. The linear ranges for several flavonoids as rutin, baicalin, silybin, puerarin and complanatuside are respectively 2.0×10^-10-1.4×10^-8 mol/L, 1.4×10^-9-5.8×10^-8 mol/L, 8.2×10^-9-2.7×10^-7 mol/L, 5.0×10^-10-1.3×10^-7 mol/L and 3.8×10^-9-2.7×10^-7 mol/L. The method could be used to determine the total efficacy of flavone in propolis. It was evaluated for 4.16% of rutin in the propolis capsule product of Sino-Sci Biological Institute and very closed to that of labeled content of 4.30%.
Keywords Electrochemiluminescence; Luminol; Quenching; Flavonoids; Propolis

1. INTRODUCTION
The flavonoids are the important effective components of many medicinal herbs and nutritional foods. Their clinical and health care efficacy were partly attributed to their natural reductibility to reduce or eliminate the superoxide anion radicals in human body^[1]. How to determine the contents or efficiency of flavonoids in those raw materials and processed products is a challenge for the development and quality controlling of those medical and health care products. The more sensitive methods to determine the content of flavonoids other than the methods such as chromatographic analysis^[2] or spectrum analysis^[3] are expected.
    The electrochemiluminescence(ECL) is a sensitive method for the analysis of some chemical species^[4]. It is in prospect to be used in trace analysis, immunoassay, biochemical analysis and some other fields^[5,6]. Based on the studies of electrochemiluminescence of luminol ^[7,8], in neutral medium^[9], and the great enhancement from iodide^[10], the authors have developed a sensitive method of quenching ECL to determine the flavonoids. The ECL luminous intensity linearly responded to the concentration of flavonoids such as complanatuside, silimarin, puerarin, baicalin or rutin. The method can be used to determine the total efficacy of flavonoids in propolis.

2. EXPERIMENTS
2.1 Instruments and Chemicals
A BAS-100A electrochemical analyzer (Bioanalysis System Inc., USA) was used as a potentiostat with a XFD-8C ultralow frequency signal generator (Ningbo Radio Factory, P.R.China) which provided a pulse signal. A PMT-II faint light meter, made in our laboratory, worked for determing the luminous intensity of ECL. The schema of whole installation was presented in Fig. 1.

Fig.1 The schema of installation of the ECL system

    Luminol was purchased from Fluka. The flavonoids are the products of Chinese Institute of Biological Product Identification. The propolis capsule is the product of Sino-Sci Biological Institute of Beijing. Other chemicals are all analytical reagents and water used in all experiments is the twice- distilled water, which was prepared by a quartz apparatus.
    Three-electrode system was used in all experiments. The working electrode and the auxiliary electrode were made of platinum. The working electrode was first polished with Al₂O₃ powder and then washed with 0.1mol/L NaOH and water in ultrasonic tank. A silver wire worked as the reference electrode.
    An ECL cell was prepared in advance. It was covered with a silver film by the silver mirror reaction and the black paint merely reserved a hole to keep transparence for light. This hole was situated in the middle of the window of the photoelectric multiplier (PMT). The cell and the PMT were sealed in a black box.


Fig.2 The regression curve of ECL intensity upon the concentration of I^-1

2.2 The Method for the Determination of Flavonoids
The authors have studied the ECL behaviour of luminol in neutral medium. It revealed that the luminol could produce ECL in an appropriate condition but its luminous intensity was weaker than that in alkaline medium. The additive iodide could greatly enhance the ECL luminous intensity of luminol in neutral medium. It provided an excellent basis to determine the flavonoids by quenching effect for iodide enhanced ECL of luminol. The determination was carried out in the phosphate buffer solution (pH 6.7) which containing 2.0×10^-7mol/L of luminol and 2.0×10^-7mol/L of iodide.
    1.0g of propolis powder sampled from the capsules was crashed and soaked in 10mL of 70% ethanol for 48 hours to extract the flavonoids^[11]. Took 1.0mL of the extraction to dilute to 100mL, thus a sample solution exchanging to 1.0mg propolis product per milliliter could be obtained. A certain volume of this sample solution was injected into the aforementioned detecting solution and then recorded the ECL intensity. The total efficacy of the flavone in propolis products could be evaluated from the decrease of ECL intensity.
3. RESULTS AND DISCUSSIONS
3.1 The Iodide Enhanced ECL of Luminol
The ECL luminous intensity would linearly increase upon the additive amount of I^- within the concentration range from 3.8×10^-7mol/L to 2.2×10^-6mol/L (See Fig. 2, r=0.995).
    The amplitude of the pulse potential strikingly affected the intensity of ECL (See Fig. 3).

Fig.3 The relationship of ECL intensity to the pulse amplitude

    It was concluded that the electrolysis procedure at suitable potential, not higher than 1.5V, produced the cation of luminol and the iodic free radical. The luminol cation would further be excited by iodic free radical and therefore to radiate the light. The detailed discussion could be seen in our previous paper^[10].
3.2 The Quenching Effect of Flavonoids
In the aforementioned condition, the flavonoids quenched the iodide enhanced ECL of luminol. It could be attributed to the elimination of iodic free radicals by flavonoids. The determining results of those five flavonoids are showed in Table 1.

Table 1 The analytical characteristics of quenched ECL method for determination of five flavonoids

3.3 The Determination of Total Efficacy of Flavone in Propolis
The ECL of luminol-I^- system would reduce the dependence upon the additive amount of propolis sample solution. It presented a linear regression within the content range of propolis from 33.3m g/ml to 104.6m g/ml (See Fig. 4, r=0.996).

Fig.4 The linear response of ECL intensity on propolis

    It indicated that the quenched ECL could be used to determine the total efficacy of flavone components in propolis under certain conditions. Because of the complication of flavonoids in propolis, the result has to be reported in the way of referred species, usually the rutin. From the sensitivity of 18.64 relative luminous intensity units per microgram rutin, which was calculated upon its linear regression equation, the total efficacy of flavone in propolis was evaluated for 4.16%. It was very closed to that of labeled content of 4.30%.

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