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Peng X.,Institute of Biopharmaceutical | Zhang T.-T.,China Pharmaceutical University | Zhang J.,China Pharmaceutical University
Plant Cell, Tissue and Organ Culture | Year: 2015

The effects of subculture frequency on genetic variations of Tetrastigma hemsleyanum callus under a long-term tissue culture were evaluated by means of flow cytometry (FCM), inter simple sequence repeat (ISSR), and sequence-related amplified polymorphism (SRAP) markers to clarify the variation pattern and frequency of somatic variation in the course of the subculture. The effect of subculture frequency on endogenous hormone content was also assessed by Enzyme-linked immunosorbent assay to explore the correlation within the differentiation rate, somaclonal variation frequency of callus and the endogenous hormone content. Moreover, the flavonoid content in the callus of different subculture times was compared and the antitumor activity of them was evaluated by using MTT cell proliferation and cytotoxicity assay. FCM confirmed similar ploidy level in mother plant and all callus, but some changes were observed in the ISSR and SRAP patterns, of which 10.3 and 6.06 % were polymorphic, respectively. The somatic variation frequency tended to increase with the increasing subculture time in the early stage of subculture, with a peak in the 4–6 times and then kept nearly at a constant after 8 times. High IAA content may be one reason for this variation by this period. The flavonoid content and antitumor potential increased and remained at high level during the 2nd and 6th subculture. Though there was a significant decline after the 6th subculture, they still had about the same flavonoid content and antitumor potential as those of mother plant. The results suggested that the in vitro callus of T.hemsleyanum is potentially useful for pharmaceutical uses. For supplying true-to-true seedlings, germplasm conservation and further genetic transformation, subculture frequency of callus should be controlled within 4 times to obtain clonally identical plantlets. However, for the development of germplasm improvement of the species, subculture frequency should be controlled in 6–8 times to make better use of somatic variations. © Springer Science+Business Media Dordrecht 2015.

Peng X.,Institute of Biopharmaceutical | Zhang Y.-Y.,Nanjing General Hospital of Nanjing Military Command | Wang J.,Nanjing Agricultural University | Ji Q.,Institute of Biopharmaceutical
Tumor Biology | Year: 2015

Ethylacetate extract of Tetrastigma hemsleyanum (EET) has a potent antitumor activity in vitro and in vivo. However, the molecular mechanism underlying EET-induced apoptosis remains elusive. As part of our continuing studies, we investigated the apoptosis mechanism of HepG2 cells exposed to different concentrations of EET in vitro. Confocal laser scanning was used to detect the apoptotic morphological changes. Flow cytometer and inverted fluorescence microscope were used to detect the mitochondrial membrane potential and cytosolic Ca2+ level. Western blotting analysis was used to evaluate the expression of the apoptosis-related proteins. Annexin V/PI staining was used to investigate cell apoptosis. Spectrophotometry was used to detect the activity of caspase family. The results showed that distinct apoptotic morphological changes occurred in HepG2 cells treated by EET. EET caused collapse of mitochondrial membrane potential, elevation of cytosolic Ca2+ level, and evoked release of cytochrome c from mitochondria in a concentration-dependent manner. The apoptosis was accompanied by a significant activation of caspase-3, caspase-9, and the cleavage of poly (ADP-ribose) polymerase, but there was no significant change in either the activity or the expression level of caspase-8. Furthermore, EET-induced apoptosis could be inhibited by caspase-9 inhibitor Z-LEHD–FMK but not by caspase-8 inhibitor Z-IETD–FMK. Taken together, these overall results demonstrated that EET-induced apoptosis of HepG2 cells was mediated by the mitochondrial caspase-dependent intrinsic pathway rather than the death receptor/caspase-8-mediated signaling route. © 2015 International Society of Oncology and BioMarkers (ISOBM)

He J.Y.,Institute of Biopharmaceutical | Ye X.Y.,Lishui University | Ling Q.Z.,Institute of Biopharmaceutical | Dong L.H.,Institute of Biopharmaceutical
Advanced Materials Research | Year: 2014

The production of laccase by solid-state fermentation (SSF) using Armillariella tabescens was studied. Wheat bran was selected to be the most suitable solid substrate. Several operational variables including nitrogen source, moisture content, copper and aromatic inducers were investigated. The results showed that the complex nitrogen sources, NH4NO3 coupled with peptone was shown to be the best nitrogen source. 75% of initial moisture content was proved to be appropriate. Copper significantly influenced the laccase production and the yield of laccase was improved by addition of 1.5 mM copper sulphate in the medium. Guaiacol efficiently induced the laccase production and the enzyme yield (24500U/g) was enhanced by 32% compared with he control without any aromatic inducers. Efficient production of laccase from A. tabescens can be achieved by solid-state fermentation. © (2014) Trans Tech Publications, Switzerland.

Peng X.,Nanjing Agricultural University | Peng X.,Institute of Biopharmaceutical | Peng X.,Ningbo institute of Microcirculation and Henbane | Zhuang D.-D.,Nanjing Agricultural University | And 5 more authors.
Tumor Biology | Year: 2015

Tetrastigma hemsleyanum, a rare and endangered medicinal plant, has attracted much attention due to antitumor and immunomodulatory activities. In this study, the effect and mechanism of ethyl acetate extract from T. hemsleyanum (EET) on cell cycle and apoptosis in human hepatoma HepG2 cells were investigated. Twenty-five to 200 μg/mL of EET were found to have the antiproliferation effect toward HepG2 cells determined by MTT assay. The morphology of EET-treated HepG2 cells showed evidence of apoptosis that included blebbing and chromatin condensation, nucleic fragmentation, and so on. The DNA laddering assay confirmed that DNA fragmentation had occurred during late apoptosis. The cell-cycle analysis indicated that EET was able to induce S phase arrest and typical subdiploid peak in a dose- and time-dependent manner. The apoptosis rate of 200 μg/mL treatment for 24 h was 42.24 ± 4.90 %. The protein expression of Bax and P53 was increased after treatment, while that of Bcl2 was significantly decreased in a dose-dependent manner, which suggested that a high Bax/Bcl2 ratio and an upregulated P53 might contribute to the pro-apoptotic activity of EET via the mitochondria-dependent pathway. The protein expression of cyclin-dependent kinase 1 (CDK1) was decreased in EET-treated HepG2 cells, suggesting that EET evoked S phase arrest possibly through the downregulation of cyclin A-CDK1 complex. In conclusion, the cytotoxicity on HepG2 cells induced by EET is a result of both cell-cycle arrest and apoptosis. Thus, it may have therapeutic potential for the treatment of liver cancer. © 2015, International Society of Oncology and BioMarkers (ISOBM).

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