Abstract
The conditions of heating, ionic liquid-based ultrasonic-assisted extraction combined with reverse-phase high performance liquid chromatography were optimized to simultaneously isolate and determinate brazilin and protosappanin B in Caesalpinia sappan. Ionic liquids, including [BMIM]Br, [BMIM]BF4, [BMIM]PF6 and [HMIM]PF6, were selected as extraction solvents while methanol, acetone, acetonitrile, ethanol and water were selected as dispersants. The chromatographic column was Purospher star RP-C18 (250 mm × 4.6 mm, 5 μm), a mixture of methanol and 0.2% phosphoric acid–water was used as mobile phase at a flow rate 0.65 mL/min. The result displayed that the extraction yields of brazilin and protosappanin B were highest when the concentration of [BMIM]Br methanol solution as extraction solvent was 0.5 mol/L and the solid–liquid ratio was 1:50 (g/mL). Under the optimal extraction conditions, the contents of brazilin showed a good linearity (r = 1.0000) within the range of 1.25–7.50 μg with the average recovery of 99.33%, the contents of protosappanin B also showed a good linearity (r = 0.9999) within the range of 0.50–3.00 μg with the average recovery of 98.31%. This experiment, which adopted environmentally friendly reagent as extraction solvent, not only improved the extraction efficiency, but also avoided the environmental pollution caused by organic solvent. Moreover, it was simple and reliable, and can be of important significance in the study of Traditional Chinese Medicine active ingredient extraction methods. The antibacterial activities of the ionic liquids and methanol extracts were determined using the paper disc diffusion method. The ionic liquid extract was found to possess antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MIC value of 37.5 mg crude drug/mL), β-Lactamase producing S. aureus (MIC values of 18.8 mg crude drug/mL), but not against E. coli, Extended spectrum β-Lactamases E. coli and P. aeruginosa. Compared with the ionic liquid extract, the methanol extract was found to have antibacterial activity against S. aureus and methicillin-resistant S. aureus (MIC value of 75.0 mg crude drug/mL), β-Lactamase producing S. aureus (MIC values of 150.0 mg crude drug/mL). However, the same, the methanol extract did not have antibacterial activity against E. coli, Extended spectrum β-Lactamases E. coli and P. aeruginosa.
Highlights
Caesalpinia sappan L. (C. sappan) belonging to family Caesalpiniaceae, an important source for pink and bright reddish-purple colors, has been cultivated in South-East Asia for the production of red dye, which is obtained from its heartwood
Brazilin and protosappanin B are two major bioactive homoisoflavones isolated from C. sappan, which are used as the marker components for the quality control of the crude drug in the current version of China Pharmacopoeia [11]
Linear relationship study According to the chromatographic condition and the sample processing method, a series of the mixed stock solutions with the injection volumes of 2, 4, 6, 8, 10 and 12 μL were analyzed by high-performance liquid chromatography (HPLC)
Summary
Caesalpinia sappan L. (C. sappan) belonging to family Caesalpiniaceae, an important source for pink and bright reddish-purple colors, has been cultivated in South-East Asia for the production of red dye, which is obtained from its heartwood. (C. sappan) belonging to family Caesalpiniaceae, an important source for pink and bright reddish-purple colors, has been cultivated in South-East Asia for the production of red dye, which is obtained from its heartwood. Pharmacological researches have shown that C. sappan possesses a wide range of pharmacological activities, including antibacterial [2, 3], antioxidant [4, 5], anti-inflammatory [6, 7], anticarcinogenic [8, 9] and apoptosis-inducing activities in Hela cells [10]. To date, few high-performance liquid chromatography (HPLC) methods for the simultaneous determination of both brazilin and protosappanin B in the crude drug have been reported in the literature [15–17], and have many shortcomings, for example, low yield, many impurities and poor quality
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