| OBJECTION1. To optimize the extraction process of UPE for salidroside, the extraction parameters including extraction rate, extraction pressure (Mpa), solid-liquid ratio (ml:g) and ethanol concentration (%) were optimized by CCD-RSM. And to compare it with reflux extraction, ultrasound-assisted extraction, and the microwave extraction, the extraction yield of salidroside and the extraction-consuming time were analyzed.2. We establish6kinds of anti-oxidant systems in vitro, and compare with the corresponding positive control in order to comprehensively evaluate Scavenging activity of extract obtained by four kinds of extraction approach and salidroside for free radical including DPPH·, O2-, OH·, ABTS·+and so on. In addition, the inhibitory activity for lipid peroxidation, reducing power, total antioxidant activity were also evaluated.3. To study the anti-tumor activity of the extracts obtained by4extraction techniques, MTT assay was applied to determine the proliferation rates of the extract obtained by 4extraction techniques for A549cells and CNE-2cells. Inverted microscope was used to observe as the morphological changes of the tumor cells in various time intervals. The experiment aims at doing a preliminary comparative study on inhibiting activity in vitro of extract obtained by four extraction techniques.Methods1. Optimization for UPE of salidroside from rhodiola rosea by CCD-RSM①Rhodiola was placed in sealed plastic bags after preprocessing. Then extraction solvent of corresponding concentration was added according to solid-liquid ratio, and introduced them to the isostatic pressure vessel according to the default operation parameters. Then solutions of crude extracts were high-speed centrifugated, and the supernatants were filtrated through0.22μm filter membrane.②The chromatographic conditions of the liquid chromatography was determined by the pre-experiment. Then the standard curve was rendered after the preparation of the standard solution of a certain concentration gradient. The sample solution was determined under the same chromatographic conditions, and the average peak area was calculated simultaneously. Finally, extraction rate of salidroside was calculated according to the standard curve.③The schemas of three-dimensional analysis and contour maps were drawn using Design-Expert8.0.0software based on regression equation. After investigated the configuration of response surface curve and evaluated the interaction between any two factors, the optimum technology was determined.④Under the same conditions, we compared UPE with the other three conventional methods on the extraction rate and extraction-consuming time to comprehensively assessed the various extraction methods.2. Determination of antioxidant activity of salidroside and the extract obtained by each extraction methods in vitro①Rhodiola was extracted by four extraction methods including ultrahigh pressure extraction(UPE), reflux extraction(RE), ultrasound-assisted extraction(UE) and microwave extraction(ME) under the predetermined experimental parameters. And the crude extracts were regard as a sample solution to be tested;②The sample was proportionately mixed with the DPPH reaction solution (accurately preparation) was measured the absorbance values in the wavelength of517nm, as well as the positive control and the negative control. Finally, the clearance rate of the sample for DPPH*radical was calculated;③The sample was first mixed with the O2-NADH-NBT-PMS solution system in proportion, then the mixture was measured the absorbance values in the wavelength of560nm, as well as positive control and the negative control to test the O2-radical scavenging activity;④To test the clearance rate of OH· free radical, the sample was reacted with the solution containing EDTA, FeC13, deoxyribose, Vc and H2O2, and then a certain amount of TBA, TCA were added during water bath. Finally, the mixture was measured absorbance values at532nm, as well as the positive control and negative control. And the clearance rate of solution was calculated;⑤ABTS·+stock solution was prepared by ABTS and potassium persulfate solution and diluted to an absorbance of0.70±0.02with ethanol at the wavelength of734nm, the sample was mixed with ABTS·+dilutions, and measured at734nm, the absorbance value of the negative control was measured at the same time, then the ABTS·+removals rate was calculated;⑥Yolk homogenate was mixed with the sample and FeS04·7H20solution was added. After that the solution was incubated for a period of time. Then TCA, TBA, acetic acid solution were added during the water bath and cooled extraction using n-butanol, finally the solution was centrifuge and the supernatant was measured at532nm; meanwhile the absorbance value of the negative control, the positive control were measured, the sample lipid peroxidation inhibition capacity was calculated;⑦Sample solution was mixed with the phosphate buffer solution, potassium ferricyanide solution and incubated in a water bath for a period of time, to terminate the reaction TCA was added, then the mixture was centrifuged. After distilled water, FeC13were added in turn, the supernatant solution was measured at700nm and Vc was used as positive control;⑧P-carotene was dissolved in the solution containing chloroform, linoleic acid, and Tween-20, and then the mixture was transferred to a round bottom flask to remove Chloroform by rotary evaporation at50℃, After that distilled water was added to make a reaction solution; certain concentration of the sample solution and the reaction solution was added to each test tube, and placed in a water bath of50℃, the mixture was measured at470nm every25minutes for a total of150minutes. Simultaneous determination of the absorbance value of the negative control and the positive control were processed and then the total antioxidant the total antioxidant capacities of the samples were calculated.3. Comparison of inhibitory action on proliferation of the four crude extracts of rhodiola in A549and CNE-2cells①Using the above four methods to extract Rhodiola. The crude extracts (UPER, RER, UER and MER) were regard as a sample solution;②A549and CNE-2cells were cultured using RPMI-1640medium and the medium was changed every two days. The cells were subcultured when they grow to degree of blend about80%;③The A549and CNE-2cells were trypsinized in logarithmic phase and suspended in a medium containing fetal calf serum, then the cells were counted and inoculated in96-well plates. Then placed in incubator for24h and original culture media was aspirate,90μl fresh medium was added. After24hours, the culture media was changed. The experimental group, positive group and negative group were respectively added10μl sample solution in different concentration,5-FU solution, and hysiological saline. Each group were culture for24,48,72and96h and repeated three times;④At each time point, the culture medium was removed, then90μl of fresh medium and10μl of MTT solution were added and incubated for4hours. The supernatant was subsequently removed and150μl of DMSO was added. After the purple crystals was fully dissolved, the mixture was measured at the wavelength of570nm and the rate of cell growth inhibition was calculated;⑤Based on Step3, the morphology of the tumor cells were observed at each time point by inverted microscope.Results1. Optimization for UPE of salidroside from rhodiola rosea by CCD-RSM The optimum parameters of the Pressure extraction method were set as follows:77.3%ethanol concentration, liquid to solid ratio of29.5, extraction pressure of255.5MPa, the maximum salidroside extract rate was about Y=9.46mg g-1; the extract rate of pressure extraction method (9.29mg· g-1) was higher than the other three extraction methods (reflux:7.10mg g-1; Ultrasound:7.72mg· g-1; Microwave:8.48mg· g-1), and the extracted time (2min) was significantly shorter than the reflux extraction(2h) and ultrasonic extraction(30min).2. Determination of antioxidant activity in vitro for extract obtained by each extraction methods and salidrosideCompared with the positive reference, UPER had he higher ability in clearing the DPPH·, O2-·, OH· radical activity and a stronger inhibition of lipid peroxidation, reducing power, total antioxidant capacity, it also had a significant capability in clearing ABTS·+, and showed dose-dependent effect; UER had strong DPPH·, O2-· scavenging ability, as well as a great total antioxidant capacity While the ability in clearing OH·, the inhibition of lipid peroxidation and the reduction capacity were weaker than UPER. MER showed moderate antioxidant activity in the six kinds vitro radical generating system; and the RER in had the weakest anti-oxidizing activity in the four extraction methods.For the salidroside monomer compounds, except for the clearing of the O2-·, OH· indicators at the maximum concentration reached50%clearance rate, IC50was missing, the other three detection indicators do not exist, and the substance Vc, BHT, TBHQ had IC50value in the five antioxidant activity detection, so the salidroside overall antioxidant activity is weaker than the three standard substance.3. Comparison of the four crude extract of rhodiolan the inhibitory effect for A549and CNE-2cell proliferation in vitroMTT assay showed that:①, The half inhibitory concentration(IC50) of the four crude extract to A549cell was2.088mg/ml,2.237mg/ml,1.974mg/ml,2.931mg/ml, so the in vitro anti-tumor activity in order as follows:MER>UPER>UER>RER. There was no significant difference in UPER, UER, MER (P<0.05), while any one of the three groups was significantly different from the RER team (P>0.05), so the A549cell proliferation inhibitory activity of UPER, UER, MER to A549cells were stronger than the RER.②The IC50of UPER, UER, MER, RER to CNE-2cell was2.239mg/ml,2.385mg/ml,2.277mg/ml,2.782mg/ml. Proliferation inhibition activity results showed that there was no significantly difference between UPER and MER (P<0.05) as well as UER and MER (P<0.05), but there was significantly difference between UPER and UER (P>0.05). And UPER, UER and MER were significantly different from RER respectively (P>0.05). Therefore, similar proliferation inhibition activity was observed among UPER, UER and MER to CNE-2cells, while the lowest activity was observed in RER.Conclusion1. Extracted salidroside from Rhodiola, UPER has the higher extraction yield and the shorter extraction time than RER and UER.2. Compared with the other three conventional extraction methods, the crude extracts we get from UPER show the highest antioxidant activity, which may be related to the less time requirement, the stronger extraction ability of antioxidant active ingredient and the less active ingredient degradation.3. UPER, UER, MER has the similar capabilities in inhibiting human non-small cell lung cancer cell A549and human nasopharyngeal carcinoma cell line, CNE-2. But compared with the RER, the anti-proliferative activity of the three groups were significantly higher. |
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