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Determination of berberine in Rhizoma coptidis using a β-cyclodextrin-sensitized fluorescence method

In this work, a method for the determination of berberine in Rhizoma coptidis using β-cyclodextrin-sensitized fluorescence technology is established. Berberine is the main extract of Rhizoma coptidis, a medicinal material, which causes an envelope reaction with β-cyclodextrin to generate fluorescence sensitization. In the environment of its own aqueous extract, with 0.0065 mol L−1 of β-cyclodextrin, a fluorescence excitation wavelength (λex) of 345 nm and an emission wavelength (λem) of 540 nm were selected to avoid interference from other distractors. The fluorescent sensor for the detection of berberine exhibits a low limit of detection (3.59 × 10−9 mol L−1) and a wide linear range from 2.7 × 10−7 mol L−1 to 2.7 × 10−6 mol L−1. Our sensor can be also used to detect berberine in real medicinal materials. The content of berberine in Rhizoma coptidis medicinal material was found to be 7.60% using this method with an average recovery rate of 99.5%. The result obtained by thin-layer chromatography with fluorescence detection was 7.61%, which is consistent with the result from the β-cyclodextrin sensitized fluorescence method. This method is simple and environmentally friendly with high sensitivity and good selectivity and gives reliable results, which is promising for practical application.


3
The solution of 0.013 mol·L -1 β-CD was prepared by dissolving 3.7500 g of β-CD in hot water, after 30 min ultrasound treatment and diluted to 250 mL with water, and diluted to appropriate concentration with water as needed.

Apparatus
Fluorescence measurements were performed on a Hitachi (Tokyo, Japan) F-4500 fluorescence spectrophotometer equipped with a xenon lamp and 1 cm quartz cell.
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. KQ-50 NC ultrasonic cleaning device was used for Ultrasonic treatment. Silicone G plate (100 mm × 200 mm, silicone thickness 0.20-0.25 mm, Qingdao Ocean Chemical Plant) was used for component separation plate.

Rhizoma Coptidis sample preparation
The Rhizoma coptidis (RC) water extraction was prepared by dissolving 0.0500 g of RC in 100℃ water for10 min and ultrasound treatment for 30 min. The mixture was filtered through a paper filter and washed several times with water. The filtrate was collected in a 100 mL volumetric flask, diluted to the mark with water and mixed well. The concentration of the water extract was expressed as 0.500 mg·mL -1 (0.500 mg RC per mL solvent).

Experimental method
Put 0.850 g β -CD in a 50 mL volumetric flask, add distilled water to dissolve and dilute to scale, and shake well. The β -CD solution was then diluted with distilled water to a concentration of 0, 2.0 ×10 -3 , 4.0 ×10 -3 , 6.0 ×10 -3 , 8.0 ×10 -3 , and 10.0 ×10 -3 mol·L -1 , respectively. 10mL of β -CD solution was placed in 6 volumetric bottles(25mL), respectively, with excessive berberine added. The solution was shaken for 72h at constant temperature (25.0 ± s0.5) ℃, centrifugated for 10min, and filtered with a 0.45 μm microporous membrane. The filtrate was diluted with distilled water and shaken well. Taking distilled water as blank, the absorbance was measured at the wavelength of 345 nm, and the concentration of berberine was calculated. Taking the concentration of β -CD solution as the x-coordinate and the concentration of berberine as the y-coordinate, the phase solubility diagram was made and the regression equation was obtained. According to the regression equation, the inclusion constant K is calculated as follows: (1) where b is the slope of the regression equation, and S 0 is the intercept.
A series of 10 mL volumetric flasks were prepared containing a certain amount of β -CD and BH solution. The mixtures were diluted to the mark with water and mixed well. After setting aside for 20 min, the two-dimensional fluorescence spectrum and three-dimensional fluorescence spectrum were measured at room temperature.
A series of 10 mL volumetric flasks were prepared containing a certain amount of β -CD, BH solution and buffer solution. The mixtures were diluted to the mark with water and mixed well. After setting aside for 20 min, the two-dimensional fluorescence spectrum was measured at room temperature.
A series of 10 mL volumetric flasks were prepared containing a certain amount of β -CD and BH solution. The mixtures were diluted to the mark with water and mixed well. After setting aside for 20 min, the UV-Vis Absorption Spectrum was measured at room temperature.
L-tryptophan (quantum yield 0.14 at excitation wavelength of 280 nm) was used as a reference in measuring quantum yield of β-CD-BH complex. For the measurement, a 1.8 mg mL -1 L-tryptophan solution and a 2.0 μg·mL -1 BH solution containing 7.5 mg·mL -1 (0.0065 mol·L -1 ) β-CD were prepared. Absorption and fluorescence spectra were measured. Quantum yield of the β-CD-BH complex was calculated by the following equation. Where Fu and Fr were the integral fluorescence intensity of unknown and reference solutions, Au and Ar were the absorbance of unknown and reference solutions at their excitation wavelengths, respectively. Before measuring the quantum yield, excitation and emission spectra were corrected 6 according to the operation manual of the F-4500 fluorescence spectrophotometer.

Table S1
The detection limits of other methods for the determination of berberine in Rhizoma Coptidis.