Isolation, Structure Elucidation, Semi-Synthesis, and Structural Modification of C19-Diterpenoid Alkaloids from Aconitum apetalum and Their Neuroprotective Activities.

Five new aconitine-type C19-diterpenoid alkaloids, apetalrines A-E (1-5), were isolated from Aconitum apetalum. Their structures were determined by analysis of 1D and 2D NMR, IR, and HRESIMS data. Semisynthesis of apetalrine B (2) from its parent compound aconorine was achieved to confirm the structure proposed. Twenty derivatives of 2 (11a-11l, 12a, 12b, 12d, 12e, 12j, 12k, 12m, 12n) were synthesized via a unified approach relying on simple coupling reactions. The evaluation of neuroprotective effects of compounds (1-5, 11b, 11c, 11f-11i, 12a, 12b, 12d, 12e, 12k, 12m, 12n) with low cytotoxicity revealed compound 2 to exhibit good neuroprotective effects in H2O2-treated SH-SY5Y cells at a concentration of 50 µM. A series of studies using flow cytometry, staining, and Western blotting on 2 indicated that its neuroprotective effects may arise from inhibiting cell apoptosis.

Diterpenoid alkaloids from Aconitum anthoroideum that offer protection against MPP+-Induced apoptosis of SH-SY5Y cells and acetylcholinesterase inhibitory activity.

Nine unprecedented diterpenoid alkaloid, including a diterpenoid alkaloid featuring a diterpenoid moiety, anthoroidine A; one bisditerpenoid alkaloid joined with a carbon-carbon single bond, anthoroidine B; three racemulosine-type C20-diterpenoid alkaloids, anthoroidines C-E; one hetidine-type C20-diterpenoid alkaloid, anthoroidine F; and three hetisine-type C20-diterpenoid alkaloids, anthoroidines G-I, together with ten known diterpenoid alkaloids were isolated from Aconitum anthoroideum DC. Their structures were established via detailed spectroscopic analyses. Most of the isolated compounds along with five known diterpenoid alkaloids obtained in a previous study were screened for neuroprotective activities and acetylcholinesterase inhibitory effects. Nominine showed potent protective activity against MPP+-induced apoptosis in SH-SY5Y cells, with a rescue rate of 34.4% (50 µM). Rotundifosine F showed a significant inhibitory activity against AChE (IC50 = 0.3 µM). The structure-activity relationship of these alkaloids is also briefly discussed.

Four new C20-diterpenoid alkaloids from Aconitum rotundifolium.

Four new C20-diterpenoid alkaloids, rotundifosines D-G (1-4), along with eight known ones (5-12) were isolated from the whole plant of Aconitum rotundifolium Kar. & Kir. The structures of the compounds were elucidated on the basis of spectroscopic analyses, including HR-ESI-MS and 1D, 2D NMR. Rotundifosine F (3) is a rare C20-diterpenoid alkaloid with quaternary ammonium salt. Alkaloids 1-4, 5, 6, 9, and 12 were evaluated for cytotoxicity against MCF-7, HCT-116 and HepG2 human cancer cell lines.

Four New Diterpenoid Alkaloids from the Roots of Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a widely used traditional Chinese medicine and an important source of clinical drugs, of which the parent and lateral roots are known as 'Chuanwu' and 'Fuzi', respectively. Four new C19 -diterpenoid alkaloids, carmichasines A - D (1 - 4), were isolated from the roots of Aconitum carmichaelii, together with twelve known compounds (5 - 16). Their structures were elucidated via spectroscopic analyses, including HR-ESI-MS, IR, and NMR. Carmichasine A (1) is the first natural C19 -diterpenoid alkaloid possessing a cyano group. Most of the diterpenoid alkaloids isolated were C19 -category, which might provide further clues for understanding the chemotaxonomic significance of this plant. The cytotoxicity of the new compounds was also investigated against several human cancer cell lines, including MCF-7, HCT116, A549, and 786-0, and none of them showed considerable cytotoxic activity.

C18-Diterpenoid alkaloids from Delphinium anthriscifolium var. majus.

Five new C18-diterpenoid alkaloids, anthriscifoltines C-G (1-5), along with four known diterpenoid alkaloids anthriscifolcines C-F (6-9), were isolated from the extract of Delphinium anthriscifolium var. majus. Their structures were elucidated by extensive spectroscopic analyses (including 1D-, 2D-NMR, and HR-ESI-MS). Compounds 1-5 were also evaluated for their cytotoxic activity against MCF-7, HepG2, and H460 human cancer cell lines.

Diterpenoid Alkaloids from Two Aconitum Species with Antifeedant Activity against Spodoptera exigua.

Twenty-five diterpenoid alkaloids were isolated from the roots of two Aconitum species. The structures of seven new C19-diterpenoid alkaloids, apetaldines A-G (1-7), and 10 known alkaloids (8-17) from Aconitum apetalum and eight known alkaloids (18-25) from Aconitum franchetii var. villosulum were elucidated via HRESIMS, IR, and NMR data. Alkaloids 1-10, 15, 16, and 18-25 were screened for their antifeedant activity. Among the compounds tested, chasmanthinine (19) showed highly potent antifeedant activity with an effective concentration for 50% feeding reduction (EC50) at 0.07 mg/cm2. The antifeedant structure-activity relationship of the diterpenoid alkaloids is also discussed.

Diterpenoid Alkaloids from Delphinium anthriscifolium var. majus.

Extensive phytochemical investigation on the whole herbs of Delphinium anthriscifolium var. majus led to the identification of fourteen diterpenoid alkaloids, including three new C20-diterpenoid alkaloids (anthriscifolsines A-C, 1-3), six new C19-diterpenoid alkaloids (anthriscifolrines A-F, 4-9), and five know compounds (10-14). Among them, anthriscifolsine A represents a novel C20-diterpenoid alkaloid characterized by a seco C-ring. The structures of the isolated compounds were elucidated by extensive spectroscopic methods, including HR-ESI-MS, X-ray, and 1D and 2D NMR experiments. Bioactivity of compounds 3-6 was evaluated for their cytotoxicity against the MCF-7, HepG2 and H460 cancer cell lines.

Five New C19 -Diterpenoid Alkaloids from Delphinium tianshanicum W.T.Wang.

Five new diterpenoid alkaloids, tianshanitines A-E (1 - 5), along with ten known compounds (6 - 15), were isolated from the EtOH extracts of the whole plant of Delphinium tianshanicum W.T.Wang. Their structures were determined based on extensive spectroscopic analyses, including 1D- and 2D-NMR, HR-ESI-MS, and the structure of tianshanitine C (3) was confirmed by X-ray diffraction analysis. Tianshanitine A (1) is the first example of natural diterpenoid alkaloid containing a benzoyl group at C(1) position. Tianshanitine B (2) is a rare natural diterpenoid alkaloid bearing a OH group at C(16) position. Compounds 1 - 5, 6, 8, 10, 12 and 14 were evaluated for cytotoxicity against HCT116, MCF-7 and HepG2 human cancer cell lines.

Potassium Hexacyanoferrate (III)-Catalyzed Dimerization of Hydroxystilbene: Biomimetic Synthesis of Indane Stilbene Dimers.

Using potassium hexacyanoferrate (III)-sodium acetate as oxidant, the oxidative coupling reaction of isorhapontigenin and resveratrol in aqueous acetone resulted in the isolation of three new indane dimers 4, 6, and 7, together with six known stilbene dimers. Indane dimer 5 was obtained for the first time by direct transformation from isorhapontigenin. The structures and relative configurations of the dimers were elucidated using spectral analysis, and their possible formation mechanisms were discussed. The results indicate that this reaction could be used as a convenient method for the semi-synthesis of indane dimers because of the mild conditions and simple reaction products.

Cyanogenetic glycosides and simple glycosides from the linseed meal.

Three new cyanogenetic triglycosides linustatins A-C (1-3), and two new simple glycosides linustatins D and E (4 and 5) were isolated from the 70% ethanol extract of flaxseed meal (Linum usitatissimum L.). Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All of the isolates showed moderate activities against aldose reductase and weak activities against α-glucosidase, DPP-IV, and FBPase at the same concentrations as the positive control drugs.

Suppression of angiogenesis and tumor growth in vitro and in vivo using an anti-angiopoietin-2 single-chain antibody.

Hepatocellular carcinomas (HCCs) are tumors with a highly developed vascular architecture. HCC cells require access to blood vessels for growth and metastasis; therefore, the inhibition of angiogenesis represents a potential therapeutic target for HCC that may reduce the mortality and morbidity from HCC. Various attempts to develop an anti-angiogenic therapy have been made in past decades; however, modest results have been achieved in clinical trials and the challenge of HCC treatment remains. Single-chain antibodies (scFv) are characterized by low molecular weight, low immunogenicity, high penetration and a short half-life, and are easy to produce on a large scale by genetic engineering. Accordingly, an scFv against a specific angiogenic regulator, such as angiopoietin (Ang), may be a promising anti-angiogenic therapy for HCC. Our previous study indicated that an imbalanced expression of angiopoietin-2 (Ang-2) vs. angiopoietin-1 (Ang-1) in HCCs contributes to initiation of neovascularization and promotes the angiogenesis and progression of HCCs. Therefore, we suggest that specific Ang-2-targeting interventions may be valuable in the treatment of HCC via remodeling the neovascular network and changing the tumor microenvironment. In this study, a prokaryotic expression vector of Ang-2 was constructed and purified human Ang-2 protein was isolated. An scFv against human Ang-2 (scFv-Ang2) was identified and purified via phage display technology, and the effects of scFv-Ang2 in vitro and in vivo on HCC in nude mice were evaluated. The results show that scFv-Ang2 inhibits vascular endothelial growth factor (VEGF) and Ang-2 induces the proliferation, migration and tubule formation of human umbilical vein endothelial cells (HUVECs) in vitro. In the in vivo assay, statistical indices, including tumor weight and volume, metastases to lungs, CD31 expression and the microvessel density (MVD) count in the scFv-Ang2-treated group of mice were significantly lower than those in the control group (P<0.05). In conclusion, the successfully generated scFv-Ang2 showed significant inhibitory effects on the angiogenesis and tumor growth of human HCC in vitro and in vivo.

Effects of Roux-en-Y operations on glucose homeostasis in obese GK rats.

AIM: To investigate the therapeutic effects of different styles of gastric bypass surgery on type 2 diabetes mellitus (T2DM) GK rats. METHODS: Twenty 6-8-week-old male GK rats were randomly divided into four groups: group A was operated by Roux-en-Y gastrojejunostomy with duodenum exclusion and stomach capacity maintenance, group B was operated by loop-type gastrojejunostomy with duodenum exclusion and stomach capacity maintenance, group C was operated by Roux-en-Y gastrojejunostomy with partial gastrectomy, and group D was operated by loop-type gastrojejunostomy with partial gastrectomy. Changes of fasting blood glucose, oral glucose tolerance test (OGTT), and insulin tolerance test (ITT) in different operations were detected. RESULTS: The operations exerted good effects on controlling blood glucose in groups A, B, C, and D. There was no significant difference between groups A and C (P > 0.05) or between groups B and D (P > 0.05), while operations in groups A and C were more effective than groups B and D (P < 0.05). On the 21st day after surgery, OGTT in animals of groups A and C was significantly improved, as indicated by a 34% reduction in the area under the curve (AUC) for blood glucose (P < 0.05 versus groups B and D); pregavage insulin levels (ng/ml) were significantly decreased in groups A and C (P < 0.05 versus groups B and D). The insulin tolerance test (ITT) confirmed impaired insulin sensitivity in groups B and D, compared with groups A and C. CONCLUSIONS: Gastric bypass surgery might be effective to treat type 2 diabetes mellitus (T2DM), and Roux-en-Y gastrojejunostomy might be more effective than other operative styles.

Antifungal and cytotoxic activities of the secondary metabolites from endophytic fungus Massrison sp.

Three novel compounds with spiro-5, 6-lactone ring skeleton has been isolated from the fermentation broth of Massrison sp. which could be isolated repeatedly from wild Rehmannia glutinosa. Psetariae oryza P-2b was applied to guide fractionation of bioactive compounds produced by Massrison sp. The molecular structures were established by a variety of one- and two-dimensional NMR experiments and the compounds with similar skeleton were reported for the first time from endophytic fungi of terraneous plant. Antifungal and cytotoxic activities of the compounds were tested, compounds 2 and 3 displayed stronger antifungal and cytotoxic activities. The compounds have the potential to be antibiotic against fungal pathogens and tumor cells.

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402/R.

AIM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2,000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhIFN-gamma, monoclonal anti-CD3 antibody, rhIL-1alpha as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5 x 10(10) of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.

[Diagnosis and treatment of esophageal fistula in anterior cervical spine surgery].

OBJECTIVE: To evaluate the causes, diagnosis, treatment and prevention of esophagocutaneous fistula in anterior cervical spine surgery. METHODS: Thirteen cases with esophagocutaneous fistula in anterior cervical spine surgery were studied. RESULTS: The causes includes: (1) During the operation, esophagus was oppressed by a clasp for so long time that made a pressure necrosis of the esophagus; (2) Esophagus was injured by loose plates and screws; (3) Loose bone grafts oppressed esophagus; (4) Esophagus was injured by operative appliance in the operation; (5) Esophagus was oppressed by the plate. DIAGNOSIS: After anterior cervical spine surgery if patients had a high fever, sore throat, swelling incision, and food sediment was found in the incision, esophagocutaneous fistula should be considered. The final diagnosis could be done by esophageal radiography. TREATMENT: Fasting cure, nasogastric tube and wound drainage should be used; When the inflammation ended, patients should undergo operation of closure of the esophageal fistula. CONCLUSIONS: The esophagocutaneous fistula in anterior cervical spine surgery has several causes mentioned above. We should take precautionary measures to avoid the complication, and use appropriate treatment to cure when it happens.