Fluorescence Determination of rhubarb in the content of free anthraquinones_609

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Fluorescence Determination of rhubarb in the content of free anthraquinones_609

    Fluorescence Determination of rhubarb in the content of free anthraquinones

     Author: Wang Peiqi, Cheng-wei, GUO Xing-jie, FAN


     Abstract Objective fluorescence spectrophotometry, was established rhubarb anthraquinones in the free determination method. Method using acetone as the solvent, the λex = 460 nm

    and λem = 540 nm fluorescence intensity was measured. The results measured free anthraquinone content in 0.25 ~ 3.0 μg

    / ml showed good linearity within the relationship, linear

    regression equation is F = 122.81C 46.224 (r = 0.999 6), the minimum detection limit was 0.25 μg / ml, showed that The

    method of high sensitivity, good reproducibility, and easy to use. Conclusion The study for measuring the active ingredient

    in free rhubarb anthraquinones provides an effective and reliable analysis method.

     Key words rhubarb; free anthraquinone; fluorescence spectrophotometry

     Fluorescence Spectrophotometry for Free Anthraquinones in Rheum emodi

     Abstract: ObjectiveWith the fluorescence

    spectrophotometry, to build up free anthraquinones in Rheum emodi method of Assay.MethodsTake acetone as solvent agent, with the λex = 460 nm and λem = 540 nm determination

    fluorescence strength. ResultsFree anthraquinone contents were

    in 0.25 ~ 3.0 of μg / ml with good linear relationship. The linear equation F = 122.81C 46.224 (r = 0.999 6), the lowest detectability was 0.25 μg / ml. The method was sensitive and reproducible.ConclusionThis research for measuring live

    composition of free anthraquinone in Rheum emodi Wall provides a kind of effectively dependable analytical method.

     Key words: Rheum emodi; Free anthraquinone; Fluorescence spectrophotometry

     Active ingredient content is an important indicator of

    the quality evaluation of medicinal herbs, rhubarb is now reported detection of free anthraquinones are mainly colorimetry, thin-layer chromatography (TLC) method, high performance liquid chromatography (HPLC), micellar electrokinetic capillary Swimming chromatography (MECC) and

    capillary electrophoresis chromatography (CEC). Chromatography separation usually requires a longer analysis time, compared to the rapid spectral method has obvious characteristics; the same time, fluorescence spectrophotometry can be used to trace

    drugs or constant material analysis, and has high sensitivity, selectivity, sample less and the method is simple, etc., so in the drug quantitative detection of the active ingredient of Chinese medicine in particular, on the increasingly wide range

    of applications. Fluorescence Spectrophotometry has been the use of traditional Chinese medicine Cassia Anthraquinone determination of the total reported [1]. In this study, the use of hydroxy-anthraquinone fluorescence properties, in acetone solution, the establishment of a free anthraquinones in Rhubarb new method for fluorescence.

     An instrument and reagent

     1.1 Instrument electronic balance (Shanghai Precision Scientific Instrument Co., Ltd. FA1004-type), KQ-500B

    ultrasonic device (Kunshan Ultrasonic Instrument Co., Ltd.), LD4-2-type low-speed centrifuge (Beijing Medical Centrifuge Factory), RE-52A rotary evaporator (Shanghai Zhengrong

    Biochemical Instrument Factory), 970CRT fluorescence spectrophotometer (Shanghai Precision Instrument Co., Ltd.).

     1.2 Materials 1,8 dihydroxyanthraquinone reference

    substance (Chinese medicines and biological products), 95% ethanol (Shenyang Chemical Plant Dongling District Red Sun), acetone (Shengsen Reagent Co., Ltd.) were AR, Rhubarb Chinese medicinal herbs (herbs were purchased from Jinzhou City

Corporation), water for the sub-boiling high-purity deionized


     2 Methods and Results

     2.1 Determination of the wavelength selection by 2 ml 3.1 μg / ml of 1,8 dihydroxyanthraquinone standard solution at

    300 ~ 600 nm wavelength range for excitation wavelength scanning, the maximum excitation wavelength of anthraquinone (λex) for 460 nm, fixed The excitation wavelength, emission spectrum was scanned, by their emission wavelength (λem) to

    540 nm. The results shown in Figure 1.

     Figure 1 Anthraquinone standard solution excitation wavelength and fluorescence wavelength (abbreviated)

     Select the maximum excitation wavelength and maximum emission wavelength (ie, λex / λem = 460 nm/540 nm) as a

    follow-up determination of working conditions.

     2.2 Preparation of standard curve precision Weigh 105 ?

    drying to constant weight of 1.8 dihydroxyanthraquinone 2.5

    mg purchase 50 ml brown volumetric flask, add acetone dissolved and diluted to the scale, shake control dope too. Precise control the amount of take home dope

    0.05,0.1,0.2,0.3,0.4,0.5,0.6 ml respectively 10 ml brown flask in constant volume with acetone to acetone reagent blank, in the λex = 460 nm and λem = 540 nm, the fluorescence

    intensity measured solution, results show that the solution of the fluorescence intensity (IF) and the solution concentration (C) in 0.25 ~ 3.0 μg / ml showed good linearity within the relationship. Figure 2.

     Figure 2 fluorescence intensity of free anthraquinones

    standard curve diagram (omitted)

     2.3 The terms of investigation

     2.3.1 Solvents influence the choice of acetone, methanol, chloroform reagent to study the impact of the reaction system results show that the sample in acetone fluorescence

    intensity, high sensitivity, and the blank solvent in this

    context is almost non-fluorescent absorption ( consistent with the reported in the literature [2]), in the excitation

    wavelength of 460 nm, emission wavelength at 540 nm, the Raman small little influence on the experiment, images are more intuitive, so this experiment use of acetone as solvent, Figure 3.

     Figure 3, acetone, methanol, chloroform fluorescence effect diagram (omitted)

     2.3.2 time on the free anthraquinone fluorescence intensity according to analytical procedure, measured at different times after the placement of 1,8 - dihydroxy

    anthraquinone relative fluorescence intensity. The results in Table 1. As can be seen from Table 1, with the extension of storage time, the relative fluorescence intensity gradually decreased the fluorescence intensity in the 0 ~ 3 h to basically stable, and therefore should ensure that within 3 h measured fluorescence. Reposted elsewhere in the paper for free download http://

     Table 1 time on the free anthraquinone fluorescence intensity (omitted)

     2.4 Determination of Sample

     2.4.1 Preparation of sample solution taken 60 ? Celsius

    dried 1 h rhubarb meal 80 mg, plus 50 ml 95% ethanol, first in the 25 kHz frequency ultrasound 20 min, then heated to the micro-boiling water bath, reflux 1.5 h , using 3 000 r / min rotational speed of centrifuge 30 min, the supernatant, residue plus 50 ml 95% ethanol solution refluxed 30 min,

    centrifuged supernatant, merging the two supernatant, concentrated to shake off ethanol, plus second residue distilled water 10 ml, 1 mol / L NaOH 2 drops, chloroform 15 ml, aqueous solution after separation Yu Bing in a water bath and add diethyl ether 10 ml, hydrochloric acid 0.4 ml immediately after the sealing Zhenyao 3 min, static stratified, so three times extraction method combined ether solution, waving to make ether added 50 ml acetone, may sample solution.

     2.4.2 Precision Precision drawing experiments were 1 ml sample solution was 5 10 ml brown volumetric flask, add

acetone fixed volume. In λex / λem = 460 nm/540 nm

    fluorescence intensity measured under. The results in Table 2. RSD 0.3%, indicating good precision of the method.

     Precision of experimental results in Table 2 (omitted)

     2.4.3 Reproducibility experiment precision that take rhubarb meal 80 mg 5 copies, according to the preparation of sample solution prepared under the sample solution, in the λ

    ex / λem = 460 nm/540 nm fluorescence intensity measured under. The results in Table 3. RSD 1.5%, indicating good reproducibility of the method.

     Reproducibility of experimental results in Table 3 (omitted)

     3 Discussion

     Anthraquinone nucleus is a long-conjugated system of the

    compound, and the structure of free anthraquinones have multiple substituents-OH, adds to the π-electron conjugated

    molecular level, so a strong fluorescence intensity; as free anthracene quinone in acetone fluorescence intensity of

    greater stability, non-interference, so the text selected

    experimental determination of acetone solvent.

     The results show that the use of fluorescence spectrophotometry of free rhubarb anthraquinone content, with sample volume is small, simple, rapid and sensitive, and can be used in human serum, urine free anthraquinone of rhubarb in the quantitative analysis.


     [1] Qi Dengfu. Fluorimetric Determination of aloe-

    emodin Chinese medicine [J]. Shandong Institute of Technology,

    2002,16 (2): 5.

     [2] Yang Li-Yan, Hui-Yun Lang. Fluorescence Determination

    of total anthraquinone content in traditional Chinese medicine [J]. Journal of Analytical Science, 2004,20 (5): 551. Reposted elsewhere in the paper for free download http://www .

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