Cardiac Glycosides from the Seeds of Thevetia peruviana.

Investigation of the seeds of Thevetia peruviana resulted in the isolation of 15 new (2-16) and 18 known (1 and 17-33) cardiac glycosides. Eight 19-nor-cardenolides (1-8), including two rare 19-nor-10-hydroperoxycardenolides, were obtained from T. peruviana for the first time. All the structures were characterized by NMR spectroscopy and chemical derivatization. The inhibitory effects of cardiac glycosides 1-33 against three cancer cell lines (human lung cancer cells, P15; human gastric cancer cells, MGC-803; and human pancreatic cancer cells, SW1990) and one normal hepatocyte cell line, LO2, were evaluated, and a preliminary structure-activity relationship is discussed. In addition, cardiac glycosides 1, 22, 26, and 28 were evaluated for their apoptosis-inducing activities in MGC-803 cells, showing IC50 values in the range 0.02-0.53 µM.

Cardiac glycosides from the seeds of Thevetia peruviana and their pro-apoptotic activity toward cancer cells.

Phytochemical investigation of the seeds of Thevetia peruviana resulted in the isolation of seven cardiac glycosides (1-7), including two new compounds (1 and 2). Cytotoxicity of them toward cancer cell lines P15 (human lung cancer cell), MGC-803 (human gastric cancer cells), SW1990 (human pancreatic cancer cells), and normal hepatocyte cell LO2 suggested that compound 1 could selectively inhibit the proliferation of cancer cell lines with IC50 from 0.05 to 0.15 µM. Pro-apoptotic activity revealed that it induced the apoptosis of MGC-803 cancer cells in a dose-dependent manner. Meanwhile, treatment of MGC-803 cancer cells with 1 resulted in diminution of pro-caspases 3 and 9 and activation of caspases 3 and 9, while it increased the Bax/Bcl-2 ratio in a dose-dependent manner. These meant that 1 induced the apoptosis of cancer cells by involving the intrinsic apoptotic pathway. In addition, the cell cycle distribution of MGC-803 cancer cells treated by 1 revealed that it could lead to cell cycle arrest at the G2/M phase. Altogether, this study suggested that compound 1 may exhibit anticancer activity by its capability of induction of intrinsic apoptosis and cell cycle arrest at G2/M phase.

[Study on anti-proliferation activity and the mechanisms of alkaloid monomers from Gelsemium elegans on HepG2 cell in vitro].

OBJECTIVE: To investigate the effects of alkaloid monomers from Gelsemium elegans on proliferation of HepG2 cell in vitro and the possible mechanism. METHODS: MTT assay was used to measure the inhibitory of three alkaloid monomers on HepG2 cell in vitro. The most effective fraction was chosen to test whether the effect was in time-and dose-dependent manner. The morphological changes were observed by the light microscope and the cell cycle alteration through the flow cytometric assay. The activity of Caspase-3, Caspase-8 and Caspase-9 were detected by a Caspases colorimetric assay kit. RESULTS: The results showed that koumine, Gelsemine and Gelsenicine could significantly inhibit the proliferation of HepG2 cell and Gelsenicine, the most effective fraction, was clearly in dose- and time-dependent manners, while exhibited low cytotoxicity to the Vero cell. The cell treated with Gelsenicine for 48 h showed distinctive morphological changes. The cells treated with 200 and 400 microg/mL shrinked and fell off from the bottom. At the same time, the cells were arrested at S phase and the apoptosis increased apparently. The activity of Caspase-3, Caspase-8 and Caspase-9 was increased in a dose-dependent manner. CONCLUSION: Three alkaloid monomers from Gelsemium elegans, especially Gelsenicine, could inhibit proliferation of HepG2 cell obviously. The mechanism may be related to cell cycle arrest and activation of Caspase-3, Caspase 8 and Caspase-9.

[Changes of neuronal nitric oxide synthase-immunopositive neurons in the central amygdaloid nucleus of spontaneously hypertensive rats].

OBJECTIVE: To observe the changes of neuronal nitric oxide synthase (nNOS)-immunopositive neurons in the central amygdaloid nucleus of spontaneously hypertensive rats (SHRs). METHODS: Thirty SHRs and 30 Wistar-Koyto rats (WKYs) were sacrificed at the ages of 90, 180, and 360 days respectively to observe the changes of nNOS-immunopositive neurons with ABC immunocytochemical assay. RESULTS: No significant changes were observed in the blood pressure of WKY rats at the specified time points, when SHRs maintained significantly higher blood pressures from 20.8+/-1.1 and 26.3+/-1.0 kPa (P<0.05), gradually increasing during the development of hypertension. The nNOS-immunopositive neurons in the central amygdaloid nucleus were of moderate sizes with long intersected nerve fibers. No significant changes were found in WKY rats. The number of the positive neurons decreased with age in SHRs, especially obvious at 360 days (P<0.05), which was significantly different from that in the WKYs (P<0.05). CONCLUSION: The reduction of nNOS-immunopositive neurons in the central amygdaloid nucleus of SHRs might accelerate the development of hypertension by modulating the cardiovascular function and sensory transmission.

Changes of neuronal nitric oxide synthase-immunoreactive neurons in the paraventricular hypothalamic nucleus of diabetic rats.

OBJECTIVE: To explore the role of nitric oxide in the development of diabetes mellitus by observing the changes of neuronal nitric oxide synthase (nNOS)-immunoreactive (nNOS-ir) neurons in the paraventricular hypothalamic nucleus (PVN) of diabetic rats. METHODS: Diabetic rat models were established by means of intraperitoneal streptozotocin injections. At the end of 2, 7 and 12 weeks after model establishment, tissue sampling was performed and the number of nNOS-ir neurons in PVN were counted and quantitatively analyzed. RESULTS: The number of nNOS-ir neurons in PVN of diabetic rats remained normal 2 weeks after model establishment (P >0.05) but was significantly increased by the time of 7 weeks (P <0.05); at the 12th weeks, the number of nNOS-ir neurons of diabetic group was still greater than that of the control group (P <0.05). CONCLUSION: The number of nNOS-ir neurons in PVN increases significantly in middle and advanced stages of diabetes, suggesting the possible relations between the nNOS activity changes in PVN and the changes in cerebral neuroendocrine and adrenal activity in diabetic condition.

Protection of feruloylated oligosaccharides from corn bran against oxidative stress in PC 12 cells.

Feruloylated oligosaccharides (FOs) were prepared by autoclaving corn bran in oxalic acid (0.6%) solution, and their protection effects against oxidative stress in pheochromocytoma cells (PC 12) cells were investigated. The FOs samples, which comprised a mixture of feruloylated mono- and dipentoses with 4.88% bound ferulic acid (FA), as well as xylose, arabinose, galactose, and glucose amounting to 46.43, 40.46, 3.76, and 8.68% of the total sugars, respectively, were prepared by autoclaving the pretreated corn bran in 0.6% oxalic acid and then further separated. Antioxidant activity was tested by 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) scavenging and oxygen radical absorbance capacity (ORAC) methods. Oxidative stress was induced by H2O2 in PC 12 neuronal cell culture model. The results showed that FOs exhibited higher antioxidant activity than free ferulic acid, with an IC50 value of 11 versus 128 µM for DPPH and an ORAC value of 4.77 versus 2.62 µmol Trolox/µmol. Tetrazolium blue assay showed that the addition of FOs with an FA concentration >50 µM significantly increased cell viability after treatment with H2O2. Flow cytometry analysis showed that the addition of FOs at concentrations of 800, 200, and 50 µM significantly decreased the apoptosis rate at the sub-G0 phase from 37.5 to 12.7, 16.2, and 20.9% (P < 0.01), respectively. FOs also significantly decreased the malonic dialdehyde content and lactate dehydrogenase (LDH) activity, but increased superoxide dismutase activity in PC 12 cells treated with H2O2 and prevented the damage of cellular membranes by decreasing the release of LDH to the cultures. The addition of FA at 800 µM showed an effect similar to that of FOs at 200 µM. Therefore, the FOs prepared from corn bran are potential functional ingredients for protection against oxidative stress.

Retrograde transport of a traditional Chinese medicine, alpha-trichosanthin, and its selective neural toxicity.

OBJECTIVE: To investigate the peripheral neuronal toxicity of a traditional Chinese medicine, alpha-trichosanthin (TCS). METHODS: TCS and rhodamine-conjugated TCS were separately injected into the rat sciatic nerve. Saline and rhodamine were used alone and separately as control solutions. The motor neurons in the spinal cord and sensory neurons in the dorsal root ganglia were separately counted. The entry of TCS molecules into neurons was observed under the fluorescence microscope. The glial reactions were studied by lectin staining and immunohistochemical method. The muscles innervated by the sciatic nerve and distal to the injection sites, and the nerves proximal to the injection sites were also collected and examined. RESULTS: TCS was taken up and transported by peripheral axons, and at a dose of 1 nmol, killed more than 90% of the motor neurons in 5 days, but only one-third of the sensory neurons of the injected nerve. The loss of neurons was permanent, while the increase of glial activities was mild and transient. CONCLUSION: TCS is retrogradely transported by axons of the injected nerve. TCS shows a selective neurotoxicity on different types of neurons. Hence TCS is useful in producing neural lesion in research, and this use may also be of applicational value in treating chronic spasticity, hyperalgesia, and pain.

[Distribution of neuronal nitric oxide synthase-immunopositive neurons in rat corpus striatum and their ultrastructures].

OBJECTIVE: To observe the distribution of neuronal nitric oxide synthase (nNOS)-immunopositive neurons in rat corpus striatum and their ultrastructural features. METHODS: Brain tissue specimens were obtained from normal SD rats, in which nNOS-immunopositive neurons were visualized by ABC immunocytochemistry and observed under immunoelectron microscope with pre-embedding staining. RESULTS: Under light microscope, nNOS-immunopositive neurons appeared brown with distinct profiles of the cell body and processes. These neurons, mostly medium-sized and small cells, were located mainly in the lateral region of the corpus striatum. Only a few immunopositive neurons were detected in the medial region of the corpus striatum. Immunohistochemistry and transmission electron microscopy identified the nNOS-immunopositive neurons as interneurons possessing large nuclei with small amount of cytoplasma. The immunopositive granules were visualized as black plaques, and the larger ones distributed mainly in the cell bodies, some with monolayer membrane encapsulation. The small granules did not have the encapsulation, scattering in perinuclear regions and under the cell membrane, but not in the cell body. The immunopositive granules were also found in the axons and dendrites, but not in the vesicles of the synapses. In addition, many immunopositive terminals were found close to the blood vessels. CONCLUSIONS: nNOS-immunopositive neurons in rat corpus striatum are mainly medium-sized and small cells as is typical of the interneurons. The immunopositive granules locate in the cytoplasma, axons and dendrites, and larger granules have membrane coating while small ones do not, possibly in relation to their functions.