Studies On Ethanol Precipitation And Countercurrent Extraction Refining Processes Of Lonicerae Japonicae Flos And Artemisiae Annuae Herba Water Extract

Ethanol precipitation and countercurrent extraction, the widely applied technologies to separate impurities and active ingredients, are usually the key processes in the manufacturing of traditional Chinese medicine injections. These two refining units may significantly impact on the downstream processes and the quality of final drugs. Lonicerae Japonicae Flos and Artemisiae Annuae Herba are two of the prescription medicines of Reduning injection. Their purified extract preparation is very important for the quality of finished products. In this paper, Lonicerae Japonicae Flos and Artemisiae Annuae Herba water extract was used as sample to investigate its refining processes of ethanol precipitation and countercurrent extraction, mainly focusing on identification and optimization the critical factors for quality control. The main contents are summarized as follows:1. An efficient and sensitive HPLC method was established for simultaneously determining the contents of six main active ingredients in ethanol precipitation supernatant of Lonicerae Japonicae Flos and Artemisiae Annuae Herba water extract. The method validation including the linearity, the intra-day precision, the repeatability, the stability and the recovery test had been carried out. As a result, the linearities of six compounds were obtained with r2>0.999, and the RSD of the intra-day precision, repeatability, stability, and the recovery test were all less than 2%.2. Based on comprehensive monitoring the manufacturing process of ethanol precipitation of Lonicerae Japonicae Flos and Artemisiae Annuae Herba water extract, a qualitative and quantitative method was developed to identify the critical risk factors affecting the stability of production process for quality control. Quality risk assessment tools, such as fishbone diagram or failure mode and effects analysis (FMEA) were used to identify the potential risk factors. With collecting the normal production samples from the ethanol precipitation process in random and monitoring the fluctuation of the potential risk factors simultaneously, seven quantitative models were established for describing the impact of each potential risk factor on the recoveries of six active ingredients and the dry matter removal of ethanol precipitation. Consumption of ethanol and density of water extract which had significantly affected all of the seven evaluation indexes, were regarded as the key quality control risk factors that influenced the stability of production process. In order to improve the quality control accuracy of ethanol precipitation process of Lonicerae Japonicae Flos and Artemisiae Annuae Herba water extract, the fluctuation range of these two critical risk factors should be reduced appropriately according to production requirement of pharmaceutical factory.3. Taking the recoveries and purities of six active ingredients, and the dry matter removal as evaluation indexes, effects of eight process factors on ethanol precipitation process were studied comprehensively. The result showed that water extract density, ethanol consumption, ethanol precipitation temperature, pH of water extract and initial ethanol concentration had a significant impact on the ethanol precipitation process of Lonicerae Japonicae Flos and Artemisiae Annuae Herba water extract, while stirring speed, temperatures of water extract and ethanol didn’t. The optimized ethanol precipitation conditions were listed as follows:water extract density,1.16 ± 0.01 g · mL-1; ethanol consumption,3.5 ± 0.3 mL · mL-1; ethanol precipitation temperature,0 ± 1 ℃; initial ethanol concentration,90 ± 1%.4. Three key process parameters affecting countercurrent extraction process, including dry matter content of concentrated liquid, consumptions of hydrochloric acid and extraction solvent were screened with fractional factorial design. In addition, central composite design and response surface method was applied to optimize the countercurrent extraction process, taking the recoveries of six active ingredients and the total saccharide removal as evaluation indexes. By using satisfactory function, the optimized countercurrent extraction conditions were obtained as follows: consumption of hydrochloric acid,6.10 g · g-1; the dry matter content of concentrated liquid,41.6%; consumption of extraction solvent,12.50 g·g-1. Under this experiment condition, the recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenin acid, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C were 48.9±0.4%,76.8±0.9%,75.3±0.8%,93.9±0.9%, 91.2±1.1% and 91.3±0.7%, respectively, and the total saccharide removal was 97.5±0.2%. The experimental results showed that countercurrent extract process could effectively enrich chlorogenic acids from 9.79% in the concentrate to as much as 54.9% in the extract.