| Extraction, isolation and purification of active components from Traditional Chinese Medicine(TCM) were the preconditions for all researches, and it is also the key problem for the modernization of TCM. There existed many disadvantages in using traditional extraction techniques such as: decocting method, impregnation method, percolation method, column chromatography and so on. Their extraction rate was low, samples were impurities and high energy consumption, and it is also can cause environment pollution, the products, which made by these traditional techniques can't eliminating the defects that Traditional Chinese patent Medicines had, and the efficacy was unstable. Therefore, In viewed of these, developed a novel extraction and purification techniques, establish efficient, fast, and low energy consumption extraction and purification technical system for TCM, provided a technical supports for the pharmacology researches and the settlement of quality, efficacy of TCM. Based on these, we took Prunella vulgaris Linn as research object, after figuring out the factors which had influence on the yield of flavonids of Prunella vulgaris by using novel extraction techniques such as Ultrasonic, Microwave and Supercritical extraction, then used High Speed Counter-Current Chromatography technique to seperated the sample which was extracted by optimized conditions. The main purpose of these was attempted to establish a novel extraction and purification technical system for TCM, at the same time provided references for the research of other TCM resources.1. The technical system for extraction of flavonids from Prunella vulgaris were developed by using ultrasonic extraction. First, through single factor experiments, the factors which may have great influence on the yield of flavonids including ethanol concentration, extraction time, extraction times, solid-liquid ratio were investigated, the result showed that: under the same of other conditions, the highest yield of flavonids conditons were: 50% of ethanol concentration, 20min extraction time, solid-liquid ratio at 1:50 and extraction for five times, respectivly. Then an orthogonal test design was applied to select the optimum extraction parameters. Then , the optimized parameters were determined namely:60% of ethanol concentration, solid-liquid ratio was 1:40, extraction time was 25 minutes, extraction times was four times, then we made a parallel verification experiment for three times, the results indicated that under the optimized conditions the yield of flavonids were 4.18%, 4.10%, 4.21%, respectively, the Relation Standard Deviation RSD=1.36%. At last, in order to estimte the extraction efficiency, we made a comparison between reflux extraction and ultrosolic extraction, which under the optimized extraction conditions, the result showed that the yield of flavonids using ultrasonic extraction was 3.68 times as high as using reflux extraction.2. Microwave extraction method were developed for the extraction of flavonids from Prunella vulgaris In this chapter. The important influence factors such as ethanol concentration, extraction time, microwave power, solid-liquid ratio were considered based on the single factor experiments, the result showed that under the same of other conditions, the highest yield of flavonids conditons were: 50% of ethanol concentration, 8min extraction time, solid-liquid ratio at 1:50 and the microvave power 300W, respectivly. Then an orthogonal test were used to optimize the conditions, Then the optimized parameters were determined namely: taking 70% of ethanol concentration as solvent, used solid-liquid ratio in 1:40, extraction 10 minutes under 200W microwave power. then we made a parallel verification experiment for three times under this optimized conditions, the results indicated that the yield of flavonids were 8.61%, 8.73%, 8.69%, respectively, the Relation Standard Deviation RSD=0.70%, the differences among them were small, which indicated that the technology was feasible. At last in order to estimte the extraction efficiency, we made a comparison with reflux extraction and ultrosolic extraction, which under the optimized extraction conditions, the result showed that the yield of flavonids using microwave extraction was 2.08 and 7.67 times as high as using ultrasonic and reflux extraction, respectively.3. Supercritical fluid extraction of flavonids from Prunella vulgaris, was performed in this chapter. A single factor experiment was applied to select the optimum extraction parameters including pressure, temperature, extraction time, 90% ethanol concentration modifier consumption, the result showed that under the same of other conditions, the highest yield of flavonids conditons were: 50℃extraction temperature, 3h extraction time, 40Mpa extraction pressure and 4.5ml/g of modifier consumption, respectivly, and then an orthogonal test design was used for optimizing the result, then the optimized conditions were determined, under the optimized conditions we make an verification experiment for three times, and we also make a comparison with reflux extraction, microwave extraction, ultrasonic extraction. The result indicated that the optimized conditions of supercritical fluid extraction of flavonids from Prunella vulgaris was 40℃of temperature, 40MPa of pressure, 2.5 hours of time, 4ml/g of modifier consumption. At last, in order to estimte the extraction efficiency, we made a comparison with reflux extraction, ultrosolic extraction and microwave extraction, which under the optimized extraction conditions, the result also showed that the extraction effect of ultrasonic and microwave were better than supercritical fluid extraction, were1.07 and 2.23 times as high as supercritical fluid extraction, respectively, whereas, the extraction effect of supercritical fluid was better than reflux extraction, it's 3.43 times as high as it. 4. In the fourth chapter we successfully used High Speed Counter Current Chromatography to establish a method of isolation and purification flavonids from Prunella vulgaris. At first the temperature,flow rate and rotation rate was investigated, which may influenced the resolution and the retention of stationary phase. Then we primary selected chloroform-methanol-water(6:8:4.v/v) as elution system according to the separation effect. Based on these, then the different injection volumes and elution modes were also considered to optimize the solvent system, then the optimized solvent system was obtained namely: primary two-phase solvent system was performed with chloroform-methanol-water(6:8:4.v/v), using upper phase as stationary phase, lower phase as mobile phase, after elution for 170 minutes the mobile phase was performed by chloroform-methanol-water(10:8:4.v/v) to continue elution, when elution times arrived at 320 minute, we changed to the first mobile phase to completed the elution. Flow rate: 2.0mL/min, rotation speed: 800rpm, temperature:30℃,detection wavelength: 254nm, injection volumes: 80mg. At last we used HPLC and MS make a primary determination of every fraction, the result indicated that under the optimized conditions, the crude extracts can yielding pure hesperidin and rutin at purities 95.7%,96.2% and four unknown components purities was 94.5%,90.1%,92.8%,96.7%, respectively. |
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