ABSTRACT
Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.
Subject(s)
Amomum , Gene Expression Profiling , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Seeds/geneticsABSTRACT
OBJECTIVE：To opt imize the extraction technology of phenolic acid from Amomum tsaoko . METHODS ：The extraction technology of phenolic acid from A. tsaoko was optimized by using Box-Behnken design-response surface methodology with ethanol volume fraction ，liquid-solid ratio and extraction time as factors ，using the total contents of protocatechuic acid and vanillic acid as response value. The optimizd extraction technology was vlidated. RESULTS ：The optimal extraction technology was as follows ：ethanol volume fraction 65％，liquid-solid ratio 4∶1（mL/g），extraction time 2.5 h. After 3 times of validation tests ， average total content of protocatechuic acid and vanillic acid were 12.32 mg/g（RSD＝0.26 ％，n＝3），average relative error of which with predicted value （12.63 mg/g）was 2.45％. CONCLUSIONS ：The optimal technology is stable and feasible .
ABSTRACT
OBJECTIVE：To establish a method for the content determination of to tal flavonoids from Amomum tsao-ko ，and to optimize the purification technology by macroporous resin. METHODS ：The content of total flavonoids was measured by HPLC. The determination was performed on Eclipse Plus C 18 column with mobile phase consisted of acetonitrile- 1％ acetic acid solution （15∶85，V/V）at the flow rate of 0.8 mL/min. The column temperature was 40 ℃，and the detection wavelength was set at 256 nm. The sample size was 10 μL. Taking the adsorption and desorption performance as indexes，6 kinds of macroporous resins were screened out by static adsorption and desorption tests ；adsorption and desorption time were investigated by static adsorption and desorption kinetics tests. Using the content of total flavonoids （calculated by rutin ）as index ，with sample concentration ，sample pH，ethanol volume fraction and elution amount as factors ，based on single factor test ，orthogonal design was used to optimize the purification technology of total flavonoids from A. tsao-ko ，and validation test was performed. RESULTS ：The linear range of rutin were 0.028-0.281 mg/mL（r＝0.999 9）. The limit of quantification was 437.5 ng/mL and the limit of detection was 109.4 ng/mL. RSDs of precision ，stability and reproducibility tests were all lower than 2％；the recoveries were 96.24％-99.75％（RSD＜2％，n＝ 6）. The comprehensive capacity of adsorption and desorption of HPD 450 macroporous resin was the most suitable ，and the best static adsorption and desorption time both were 12 h. The optimal purification technology was 1.854 4 mg/mL ； ethanol elution was 8 times of the column volume. Vertificationtests show that after optimized ，the content of total flavonoids from A. tsao-ko increased from 22.556 7 mg/g to 57.728 2 mg/g. The purity of was 2.56 and stable for the content determination. Optimal purification technology is stable and feasible ，which is suitable for purifieation of total flavonoids from A. tsao-ko .
ABSTRACT
OBJECTIVE：To evaluate in vitro antioxidant activities of 4 different polar parts of ethanol extract from Amomum tsao-ko，and to lay a foundation for the research and development of antioxidant chemical components in the plant. METHODS ： The dried fruits of A. tsao-ko were crushed ，then were hearted and reflux extracted with 95％ ethanol. The extraction fluid was concentrated by rotary evaporation and evaporated in water bath to obtain the ethanol extract. The extract was dispersed in water ， and then extracted with petroleum ether ，ethyl acetate and n-butanol organic solvents one by one. Each solvent extract was combined and the lower water phase were collected. Finally ，the petroleum ether part ，ethyl acetate part ，n-butanol part and water part were obtained，after rotary evaporation concentration and water bath evaporation. Through in vitro antioxidant activity tests ，using 2， 6-di-tert-butyl-4-methylphenol（BHT）as positive control ，DPPH radical ，superoxide anion radical scavenging ability and Fe 3+ reducing ability of different polar parts of ethanol extract from A. tsao-ko were investigated. RESULTS ：The scavenging rates of 4 polar parts of ethanol extract from A. tsao-ko on DPPH radical were all over 80％；the order of scavenging ability was ethyl acetate part＞BHT＞n-butanol part ＞petroleum ether part ＞water part. Those of the 4 polar parts to superoxide anion radical were between about 30％-40％ mostly；the order of scavenging ability was n-butanol part ＞petroleum ether part ＞water part ＞ethyl acetate part ＞ BHT；but those were weaker than their scavenging ability to DPPH r adical. The polar parts of ethanol extract also had a certain reduction ability to Fe 3+；the order of the reduction ability was n-butanol part ＞BHT＞ethyl acetate part ＞petroleum ether part ＞ water part on the whole ，but that of water part rose to the stron- gest when its concentration was 4.0 μg/mL. CONCLUSIONS： The different polar parts of ethanol extract from A. tsao-kom have certain in vitro antioxidant capacity ，but the order of antioxidant activity of different polar parts was not the same in different antioxidant activity tests ；ethyl acetate part has the 163.com strongest scave nging effect on DPPH radical ，n-butanol part has the strongest scavenging ability on superoxide anion radical and reducing ability on Fe 3+.
ABSTRACT
Bioactivity-guided fractionation of MeOH extract of the dried fruits of Amomum tsao-ko led to isolation of nine compounds (1 – 9). Their structures were elucidated by spectroscopic methods including extensive 1D and 2D-NMR, as alpinetin (1), naringenin-5-O-methyl ether (2), naringenin (3), hesperetin (4), 2′,4′,6′-trihydroxy-4-methoxy chalcone (5), tsaokoin (6), boesenbergin B (7), 4-hydroxyboesenbergin B (8), and tsaokoarylone (9). Of these, compound 8 was isolated from a natural source for the first time, which was previously reported as a synthetic product. The isolated compounds (1 – 9) were tested for their inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 macrophages. Among them, three chalcone derivatives (compounds 5, 7, and 8) and a diarylheptanoid (compound 9) exhibited significant inhibitory activity on the NO production with IC₅₀ values ranging from 10.9 to 22.5 µM.