| Yohimbine and reserpine, two of the most important Rauwolfia indole alkaloids, primarily act as anti-hypertensives, sedative agents, antivirals and anti-impotences, etc. Yohimbine and reserpine are generally isolated from the roots of Rauwolfia species, where they are in low contents. And they share the similar physicochemical properties of the indole alkaloids in many plants of Rauwolfia species. Both their low contents and similar physicochemical properties make yohimbine and reserpine difficult to be isolated and purified from Rauwolfia species. In this paper, the ion exchange fiber (IEF) was chosen as the adsorbent. The adsorption behavior of yohimbine and reserpine onto the fiber was investigated and a separation method of reserpine and yohimbine from Rauwolfia species was established. The main research contents and results were showed as follows:(1) Establishment of simultaneous determination method of yohimbine and reserpine in Rauwolfia.The contents of yohimbine and reserpine in Rauwolfia were determined by High Performance Liquid Chromatography (HPLC) equipped with reversed phase C18 column (4.6 mm×250 mm,5 μm, Hypersil-ODS2, China). The detector wavelength (UV) was 270 nm. The temperature of column was adjusted to 30 ℃. The flow rate was 0.3 mL/min. The mobile phase was composed of acetonitrile-MKP solution (20 mmol/L) with V/V ratio of 40:60. The pH was adjusted to 3 by phosphoric acid. Within the range of 2.6～208 μg/mL, the standard linear regression equations of yohimbine and reserpine were y=40521x+125860 and y=55403x-36823 (y:peak area, x:concentration, mg/L), respectively. The HPLC method was sensitive and accurate, which could be applied to simultaneously determine yohimbine and reserpine in Rauvolfia. And it provided a high-speed, convenient test to extract and separate yohimbine and reserpine from Rauvolfia.(2) Study on the adsorption properties of yohimbine or reserpine on the ion exchange fiber.The adsorption properties of yohimbine or reserpine on ZB-1 strong acid cation exchange fiber (ZB-1fiber) were investigated by scanning electron microscopy (SEM), Fourier Transform Infrared Spectrometer (FTIR) and HPLC. The surface morphologies of ZB-1 fiber adsorbing and desorbing yohimbine or reserpine were analyzed by SEM, and the SEM images had shown visually the dynamic process of the increase of adsorbate with the increase of the adsorption time on the ZB-1 fiber. The FTIR spectra of the ZB-1 fiber after adsorption emerged characteristic peaks of yohimbine or reserpine. Meanwhile, the concentrations of yohimbine or reserpine in solution decreased with the increase of adsorbate on the ZB-1 fiber surface. These results confirmed the successful adsorption of yohimbine or reserpine onto the ZB-1 fiber.(3) Study on the adsorption kinetics of yohimbine or reserpine on the ion exchange fiber.The adsorption kinetics experiments of yohimbine or reserpine on the ion exchange fiber were performed by static adsorption experiments. The kinetics data were determined by HPLC and fitted by the kinetics models. The results showed that the equilibrium was attained within 20 mins. The kinetics of the adsorption followed the pseudo-second-order model as . Under the experimental conditions of c0=160mg/L and T=303 K, the pseudo-second-order model of yohimbine and reserpine were t/qr=0.0062t+0.016 (R2=0.9975) and t/qt=0.0065/+0.0198 (R2=0.9972), respectively. The rate constants of pseudo-second-order model (k2) decreased with the increased of initial concentrations within the range of 100-300 mg/L. Arrhenius equation of yohimbine and reserpine were given as lnk2=-1730.9/T-0.018 (R2=0.9609) and lnk2=-523.05/T-4.3987 (R2=0.9537), respectively. The activation energy (Ea) was calculated respectively to be to be 14.39 and 4.35 kJ/mol, implying the adsorption of yohimbine or reserpine onto the ZB-1 fiber was rapid physisorption process. The Boyd film diffusion model fitted the experimental data better than Weber and Morris intra-particle diffusion model, indicating the film diffusion was the rate-limiting step in the entire adsorption process of yohimbine or reserpine onto the ZB-1 fiber. The rate constants of Boyd film diffusion (kB) and the effective diffusion coefficients (D) increased with the increase of adsorption temperature and stirring speed. These results showed that ZB-1 fiber possessed a good adsorption kinetics characteristic for yohimbine or reserpine.(4) Study on the adsorption thermodynamics of yohimbine or reserpine onto the ion exchange fiber.The adsorption thermodynamics experiments of yohimbine or reserpine on the ion exchange fiber were performed by static adsorption experiments. The equilibrium data were determined by HPLC and fitted by four isotherm models. The results showed that the equilibrium data could preferably be modeled with the Freundlich isotherm as for both yohimbine and reserpine. Under the experimental conditions of T=303 K and c0=160mg/L, the Freundlich model of yohimbine and reserpine were lnqe=3.3457+0.7231nce (R2=0.998) and lnqe=3.3852+0.7551nce (R2=0.975), respectively. And the rate constants (kF) of Freundlich model increased with the increased of adsorption temperature. Within the range of 283～323 K, the values of 1/n were less than 1, indicating that the adsorption of yohimbine or reserpine onto the ZB-1 fiber was a favorable process of physisorption. The thermodynamic parameters of yohimbine and reserpine were calculated to be:12.60 kJ/mol<△<14.23 kJ/mol,-3.66 kJ/mol <△G<-3.26 kJ/mol,50.35 J/(mol·K)<△S<61.81 J/(mol·K) and 4.48 kJ/mol<△H< 9.58 kJ/mol,-3.47 kJ/mol<△G<-3.28 kJ/mol,24.61 J/(mol·K)<△S<45.44 J/(mol·K), respectively. The thermodynamic parameters demonstrated that the adsorption was an endothermic, spontaneous and feasible process of physisorption within the temperature range between 283 K and 323 K. And the increase of temperature would benefit the adsorption.(5) Optimization of extraction and separation process of yohimbine and reserpine from RauwolfiaThe ultrasound extraction process parameters of yohimbine and reserpine from Rauvolfia were optimized by response surface methodology. the optimum extraction conditions for yohimbine and reserpine from Rauvolfia were found to be 120 meshes,18 solvent/solid (mL/g),47 min, pH=1. Under the optimized conditions, the average yield of validation experiments (n=3) of yohimbine and reserpine were 3.392mg/g and 0.070 mg/g, respectively, which were in good agreement with their predicted value (3.4623 mg/g and 0.0721 mg/g) by model. The relative error were 2.04% and 2.91%。The influencing factors of adsorption and desorption were investigated. The results showed that optimum conditions for the static adsorption and desorption were as the following:0.01-0.03 g yohimbine or reserpine were adsorbed in 100 mL solvent of 60% ethanol, pH=5 and the adsorbent dosage was 0.1 g. The fiber that had adsorbed the yohimbine by the static adsorption could be desorbed by the desorption solution of 40% ethanol, pH=2 (10 mmol/L HCl), and reserpine could be desorbed by the desorption solution of 80% ethanol, pH=1.3 (50 mmol/L HCl). The optimum conditions for the dynamic adsorption were as the following:The yohimbine and reserpine were adsorbed in the solution concentration of 80 mg/L by fiber column and the flow speed was 5mL/min. When the elution rate was controlled to be 5 mL/min, the yohimbine was desorbed firstly from the fiber column by the desorption solution of 40% ethanol, pH=2. And then reserpine was desorbed by the desorption solution of 80% ethanol, pH=1.3 (50 mmol/L HCl).At last, yohimbine and reserpine were extracted from Rauwolfia. The extracting solution of Rauwolfia (pH=1) was adjusted to pH=5 by 0.1mol/L NaOH, and flowed through the ZB-1 fiber column until saturation with the speed of 5mL/min. Then the ZB-1 fiber column which was saturated was firstly eluted by the desorption solution of 40% ethanol, pH=2 with the flow speed of 5mL/min, and yohimbine was attained from the eluate. Secondly, the ZB-1 fiber column was eluted by the desorption solution of 80% ethanol, pH=1.3, and reserpine was attained from the eluate. After separated and purified by the ZB-1 fiber column, the concentrations of yohimbine and reserpine in the eluate reached 23.04mg/L and 19.97 mg/L, respectively. The yohimbine and reserpine in the extracting solution of Rauwolfia were separated effectively by the ZB-1 fiber column. |
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