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Objective To investigate the change of biological characteristics after stable knockdown of the coding gene of 3-methylcrotonyl-coenzyme A carboxylase β subunit (MCCC2) expression in DU145 by lentivirus shRNA. Methods Three groups were included in this study. shNC was the control group in which MCCC2 was negatively knocked down in DU145. shMCCC2 was the experimental group in which MCCC2 was knocked down. DU145 was the blank group without any treatment. The expression of MCCC2 was assessed by Western blot and qPCR. The proliferation of DU145 cells was detected by CCK8 assay. The migration ability of DU145 was detected by transwell. The apoptosis of DU145 cells was detected by flow cytometry. Results The expression level of MCCC2 in shMCCC2 group was significantly lower than that in shNC group (0.22 ± 0.02 vs 0.61 ± 0.06, P < 0.001). The proliferation (2.24 ± 0.04 vs 3.13 ± 0.15) and migration (23.96 ± 1.85 vs 49.73 ± 0.63) of DU145 cells in shMCCC2 group was significantly lower than that in shNC group, whereas the apoptosis (12.64 ± 0.30 vs 3.68 ± 0.02) of DU145 cells in shMCCC2 was significantly higher than that in shNC group. Conclusion MCCC2 knockdown significantly inhibited the proliferation and migration, and induced apoptosis of DU145 cells, which indicated that the down-regulation of MCCC2 is correlated with the change of tumor biological characteristics of DU145 cell line and can be a potential target for the treatment of prostate cancer.
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Objective To optimize the process of ultrasonic extraction of polysaccharide in Anoectochilus roxburghii and to investigate the method of protein removal. Methods The extraction rate of polysaccharide was used as the detection index. On the basis of single factor investigation, Box-Behnken experimental design and response surface method were used to optimize the three factors of material-liquid ratio, ultrasonic time and ultrasonic extraction temperature. The five deproteinization methods including Sevage reagent method, TCA method, salt method (NaOH-CaCl2 and NaOH-NaCl) and hydrochloric acid method were investigated with the retention rate of polysaccharide and protein removal rate. Results The optimal extraction conditions of polysaccharide from Anoectochilus roxburghii were as follows: liquid-to-solid ratio was 10∶1, extraction temperature was 48 ℃ and extraction time was 36 min with extraction 2 times, ultrasonic power was 300 W, the extraction rate was 13.13%. NaOH-CaCl2 deproteinized methods∶ the loss rate of polysaccharide was 18.74%, and the removal rate of protein was 95.62%. Conclusion Ultrasonic extraction is easy to operate, and the optimized extraction method can achieve a high extraction rate. NaOH-CaCl2 deproteinization methods can get high protein removal rate and polysaccharide retention rate. This method is suitable for the research and development of the active components of the polysaccharides from Anoectochilus roxburghii.
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@#Transcriptome sequencing was performed for the first time on Anoectochilus roxburghii(AR)in different harvesting periods using RNA-seq high-throughput sequencing technique, and the results were verified and analyzed by Q-PCR and HPLC. A total of 51, 370 genes were obtained by transcriptome sequencing and annotated to the database of Nr, GO, Swiss-Prot, KEGG and KOG. The species that were sequenced according to the homology sequence were the same as AR monocotyledon plants. Through comparison of AR transcriptome in different periods, it was found that the differences were mainly in flavonoid biosynthesis-related genes. The expression levels of flavonoid biosynthesis-related genes(trans-cinnamate 4-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, flavonol synthase, shikimate O-hydroxycinnamoyltransferase and flavonoid 3′, 5′-hydroxylase)were verified by Q-PCR, and the results were consistent with those of transcriptome sequencing. The contents of 6 flavonoids(rutin, isoquercitrin, narcissin, quercetin, kaempferol and isorhamnrtin)were determined by HPLC. The results showed that the expression of flavonoid synthetic gene in AR increased with the growth time, and the variation trend of flavonoid compound content and gene expression were basically consistent. Combined with transcriptome data, the biosynthetic pathway of flavonoid content in AR was plotted. This study provides important genetic resources for the key genes of flavonoid synthesis in AR and the biosynthesis of flavonoids, as well as the basis for the development of its medicinal value.
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OBJECTIVE:To analyze volatile oil from the roots and fruits of Kadsura longipedunculata. METHODS:GC-MS method was adopted. The determination was performed on HP-5MS quartz capillary column with nitrogen as carrier gas at the flow rate of 0.9 mL/min. The temperature of sample inlet was 250 ℃,the initial temperature of column was set at 50 ℃(temperature programming). The column pressure was 80 kPa;the ratio of split sampling was 40 : 1. The sample size was 1.0 μL. Mass chroma-tography was as follows:electron bombardment ion source as ion source;electron energy of 70 eV;inlet temperature was 230 ℃;scanning range of m/z 40-400,scanning interval time of 1.0 s. RESULTS:Totally 43 chromatographic peaks were detected in vola-tile oil from the roots of K. longipedunculata,and 24 peaks were identified,accounting for 91.5%;main components were sesqui-terpenes and monoterpenoids,among which the relative contents of (+)-delta-cadinene,γ-cadinene and isovalencene were in high level. Totally 52 chromatographic peaks were detected in volatile oil from the fruits of K. longipedunculata,and 36 peaks were iden-tified,accounting for 82.2%;main components were sesquiterpenes and monoterpenoids,among which relative contents of caroph-yllene,γ-cadinene and valencene were in high level. CONCLUSIONS:The study basically confirm main volatile components in volatile oil from the roots and fruits of K. longipedunculata,and the roots and fruits of K. longipedunculata have significant differ-ence.
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In this study, a rapid and sensitive analytical method was developed for the determination of 10 major compounds (procyanidin B1, catechin, procyanidin B2, rutin, isoquercitrin, kaempferol-3-O-rutinoside, astragalin, quercitrin, quercetin, and kaempferol) in Tetrastigma hemsleyanum by using ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-MS/MS) in multiple-reaction monitoring (MRM) mode. UPLC-MS/MS assay with negative ion mode was performed on a Waters CORTECS C18 (2.1 mm x 100 mm, 1.6 μm) with the mobile phase consisting of acetonitrile (A) and 0.1% aqueous formic acid (B) in gradient elution at a flow rate of 0.25 mL · min(-1) and the column temperature was set at 45 °C. Under the optimized chromatographic conditions, good separation for 10 target compounds were obtained including chiral isomer procyanidins B1 and B2 were completely separated within 8.5 min. Satisfactory linearity was achieved with wide linear range and fine determination coefficient (r > 0.996 6), the overall recoveries were ranged from 95.44%-110.40% with the RSD ranging from 2.37%-8.69%. It is the first report about simultaneous analysis of 10 major flavonoids components in Tetrastigma hemsleyanum by using UPLC-MS/MS method, which affords highly sensitive, specific, speedy and efficient method for quality control of Tetrastigma hemsleyanum