New ψ-Santonin Derivatives from Crossostephium chinense and Their Anti-Proliferative Activities against Leishmania major and Human Cancer Cells A549.

Previously, we reported two cytotoxic ψ-santonin-amino acid conjugates isolated from the EtOAc layer of Crossostephium chinense. However, a further phytochemical investigation seems to be required because of the few reports of similar derivatives. In this study, we targeted the 1-BuOH layer, which resulted in the isolation of seven new ψ-santonin derivatives (1-7) together with ten known compounds (8-17). The structures of 1-7 were elucidated based on spectroscopic methods, including 1D and 2D NMR experiments (1H, 13C, DEPT, COSY, HSQC, and HMBC), IR spectrum, and high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS). The stereochemistry of new compounds was confirmed by NOESY and ECD calculations. All isolated compounds were evaluated by in vitro experiments for their anti-proliferative activities against Leishmania major, human lung cancer cell line A549, and Vero cells. As a result, most of the ψ-santonin derivatives, especially 1-5, showed significant cytotoxicity against L. major with a lower IC50 than the positive control we used (miltefosine).

Identification of the Toxic Compounds in Camellia oleifera Honey and Pollen to Honey Bees (Apis mellifera).

Identifying the components of Camellia oleifera honey and pollen and conducting corresponding toxicological tests are essential to revealing the mechanism of Camellia oleifera toxicity to honey bees. In this research, we investigated the saccharides and alkaloids in honey, nectar, and pollen from Camellia oleifera, which were compared with honey, nectar, and pollen from Brassica napus, a widely planted flowering plant. The result showed that melibiose, manninotriose, raffinose, stachyose, and lower amounts of santonin and caffeine were found in Camellia oleifera nectar, pollen, and honey but not in B. napus nectar, pollen, and honey. Toxicological experiments indicated that manninotriose, raffinose, and stachyose in Camellia oleifera honey are toxic to bees, while alkaloids in Camellia oleifera pollen are not toxic to honey bees. The toxicity mechanism of oligosaccharides revealed by temporal metabolic profiling is that oligosaccharides cannot be further digested by honey bees and thus get accumulated in honey bees, disturbing the synthesis and metabolism of trehalose, ultimately causing honey bee mortality.

Pharmacological Evaluation of Artemisia cina Crude CO2 Subcritical Extract after the Removal of Santonin by Means of High Speed Countercurrent Chromatography.

Artemisia species are highly important due to their economic significance as medicines, fodder and food. Artemisia cina is an endemic species to Kazakhstan. In folk medicine, water extract of A. cina was used in the treatment of bronchial asthma while the alcohol extract has larvicidal and antituberculosis activity. The most common and most extensively studied compound from this species is the terpenoid santonin. The toxicity of this compound occurs at the doses of 60 mg for children and 200 mg for adults causing among other issues xanthopsia, leading to blindness. Having this in mind, the main idea of this work was to remove santonin from the crude extract and to check if the santonin-free extract would still be of any pharmacological importance. A CO2 subcritical extract was chromatographed using high-speed countercurrent chromatography (HSCCC) for the removal of santonin. The santonin-free CO2 subcritical extract (SFCO2E) as well as the isolated compound pectolinarigenin, a flavonoid, were assessed for their pharmacological actions. From the results obtained we can safely suggest that HSCCC is an efficient methodology to completely remove santonin from the CO2 subcritical extract. It was also possible to observe promising antinociceptive and anti-inflammatory activities for both SFCO2E and pectolinarigenin at concentrations that can justify the production of a phytomedicine with this endemic plant from Kazakhstan.

The Effects of (11R)-13-(6-Nitroindazole)-11,13-Dihydroludartin on Human Prostate Carcinoma Cells and Mouse Tumor Xenografts.

BACKGROUND This study aimed to investigate the effects of the 6-nitroindazole compound and amino analog of ludartin, (11R)-13-(6-nitroindazole)-11,13-dihydroludartin (NDHL), on human prostate carcinoma cells in vitro and in mouse tumor xenografts in vivo. MATERIAL AND METHODS DU-145 and LNCaP human prostate carcinoma cells were cultured with increasing concentrations of NDHL. Cell viability was measured using the MTT assay, and cell apoptosis was measured by fluorescence flow cytometry. Mouse tumor xenografts were created by implanting 2×106 of DU-145 cells subcutaneously in the left flank. On the second day following DU-145 cell implantation, the mice in the treatment groups were injected intraperitoneally with 2, 5, and 10 mg/kg of NDHL. RESULTS Treatment of DU-145 and LNCaP cells with NDHL (range, 2.5-20.0 µM) significantly reduced cell proliferation in vitro (P<0.05). The proliferation rate of DU-145 and LNCaP cells was reduced to 27% and 24%, respectively, following treatment with 20.0 µM of NDHL. Treatment with NDHL significantly increased cell apoptosis and the formation of reactive oxygen species (ROS) formation in DU-145 cells at 48 h (P<0.05). NDHL significantly increased the proportion of DU-145 cells in the G1 phase of the cell cycle and significantly increased the expression of cyclin D1 and p21 (P<0.05). Treatment of the mice in the xenograft tumor model with NDHL significantly increased survival and suppressed tumor growth (P<0.02). CONCLUSIONS NDHL inhibited cell proliferation, increased apoptosis, and caused cell cycle arrest in human prostate carcinoma cells in vitro and inhibited mouse tumor xenograft growth in vivo.

Naturally Occurring Sesquiterpene Lactone-Santonin, Exerts Anticancer Effects in Multi-Drug Resistant Breast Cancer Cells by Inducing Mitochondrial Mediated Apoptosis, Caspase Activation, Cell Cycle Arrest, and by Targeting Ras/Raf/MEK/ERK Signaling Pathway.

BACKGROUND Sesquiterpene lactones have gained tremendous attention owing to their potent anticancer properties. The main focus of this study was to evaluate the anticancer effects of a naturally occurring sesquiterpene lactone, santonin, against human breast cancer SK-BR-3 cells. MATERIAL AND METHODS Cell counting kit 8 assay was used for the determination of cell viability. Apoptosis was detected by DAPI (4',6-diamidino-2-phenylindole) and annexin V/propidium iodide (IP) staining. Flow cytometry was used for cell cycle analysis and western blotting was used for the estimation of protein expression. RESULTS Results showed that santonin exerts significant anti-proliferative effects on the SK-BR-3 breast cancer cells in a concentration dependent manner. Santonin showed an IC50 of 16 µM against SK-BR-3 cells. DAPI staining showed that santonin caused DNA fragmentation in the SK-BR-3 cells, which is indicative of apoptosis. Annexin V/PI staining showed that apoptotic cell percentage increased up to 34.32% at 32 µM concentration of santonin. Santonin also caused an increase in the expression of Bax, caspase-3, and caspase-9, and a decrease in the expression of Bcl-2. Santonin also caused the arrest of the SK-BR-3 cells at the G2/M phase of the cell cycle and suppressed the expression of cyclin A and B1. Finally, santonin could also block the Raf/MEK/ERK pathway in breast cancer cells. CONCLUSIONS The findings of this study suggest the potential for the naturally occurring sesquiterpene lactone santonin in breast cancer treatment and also suggest that it could be developed as a promising anticancer agent.

Structure-activity relationship and synthetic methodologies of α-santonin derivatives with diverse bioactivities: A mini-review.

α-Santonin, a sesquiterpene lactone isolated from Artemisia Santonica, possesses diverse bioactivities including antioxidant, anti-inflammation, immunosuppressive, anti-roundworm, anti-malaria, etc. However, its bioactivities are not satisfactory and need to be further optimized. Thus, many α-santonin derivatives were synthesized on the basis of rings A, B and C for the discovery of new analogues with prominent bioactivities. Herein, we reviewed and discussed the related synthetic methodologies, diverse bioactivities and structure-activity relationships (SAR) of α-santonin derivatives.

Transformation of Santonin to a Naproxen Analogue with Anti-Inflammatory Activity.

Santonin, a natural product, was aromatized with molecular iodine as the catalyst. The new compound was characterized as ( S)-methyl-2-(7-hydroxy-5,8-dimethylnaphthalen-2-yl) propanoate (2) based on 2D NMR spectroscopic data. Structurally, compound 2 was highly similar to the anti-inflammatory drug naproxen. The new naproxen analogue had significant potency against cyclooxygenase 1 and 2 (IC50 = 31.0 and 66.1 µM, respectively).

A Molecular Electron Density Theory Study of the Chemoselectivity, Regioselectivity, and Diastereofacial Selectivity in the Synthesis of an Anticancer Spiroisoxazoline derived from α-Santonin.

The [3 + 2] cycloaddition (32CA) reaction of an α-santonin derivative, which has an exocyclic C⁻C double bond, with p-bromophenyl nitrile oxide yielding only one spiroisoxazoline, has been studied within the molecular electron density theory (MEDT) at the MPWB1K/6-311G(d,p) computational level. Analysis of the conceptual density functional theory (CDFT) reactivity indices and the global electron density transfer (GEDT) account for the non-polar character of this zwitterionic-type 32CA reaction, which presents an activation enthalpy of 13.3 kcal·mol-1. This 32CA reaction takes place with total ortho regioselectivity and syn diastereofacial selectivity involving the exocyclic C⁻C double bond, which is in complete agreement with the experimental outcomes. While the C⁻C bond formation involving the ß-conjugated carbon of α-santonin derivative is more favorable than the C⁻O one, which is responsible for the ortho regioselectivity, the favorable electronic interactions taking place between the oxygen of the nitrile oxide and two axial hydrogen atoms of the α-santonin derivative are responsible for the syn diastereofacial selectivity.

Selective Cytotoxicity of α-Santonin from the Persian Gulf Sponge Dysidea Avara on Pediatric ALL B-lymphocytes via Mitochondrial Targeting

Background: Acute lymphoblastic leukemia (ALL) is one of the most dominant malignancies among children, characterized by production of immature and dysfunctional blasts which are resistant to cytotoxic chemotherapeutic agents. Therefore, research protocols are currently focusing on discovery of novel anti-cancer agents to enhance survival rates and decrease unwanted side effects. Approximately two-thirds of the planet is covered by oceans with a massive range of marine organisms of interest to scientists in pharmaceutical fields. Methods: Among marine resources, sponges are known to have beneficial effects in the treatment of numerous malignancies. One fraction of crude extracts containing α-Santonin was made from the Persian Gulf marine sponge, Dysidea avara, and investigated for anticancer effects. Results: Treatment of ALL B-lymphocytes with the Dysidea avara extract caused augmentation in ROS generation, decline in mitochondrial membrane potential, mitochondrial swelling, release of cytochrome c from mitochondria and activation of caspase-3 only in mitochondria isolated from B-ALL lymphocytes. Conclusion: In brief, our results suggest that Dysidea avara extracts may selectively induce apoptosis in malignant pediatric lymphocytes.

Synthesis and biological evaluation of α-santonin derivatives as anti-hepatoma agents.

A series of α-santonin-derived compounds as potentially anti-hepatoma agents were designed and synthesized in an effort to find novel therapeutic agents. Among them, derivative 5h was more potent than the positive control 5-fluorouracil (5-Fu) on HepG-2, QGY-7703 and SMMC-7721 with IC50 values of 7.51, 3.06 and 4.08 µM, respectively. The structure-activity relationships (SARs) of these derivatives were discussed. In addition, flow cytometry and western blot assay revealed that the derivatives induced hepatoma cells apoptosis by facilitating apoptosis-related proteins expressions. Our findings suggested that these α-santonin-derived analogues hold promise as chemotherapeutic agents for the treatment of human hepatocellular cancer.

Rational drug design and synthesis of new α-Santonin derivatives as potential COX-2 inhibitors.

Sesquiterpene compounds are widely known for their numerous pharmacological activities. Herein the focus of the authors was on α-Santonin, a sesquiterpene lactone from the Artemisia genus: the aim was to determine whether α-Santonin could be considered in the treatment of inflammation and pain. To this purpose, a small series of derivatives was designed and screened in silico against the enzyme COX-2 along with the parent compound. Drug-likeness parameters were also assessed. The compounds were eventually synthesized, and few were tested to determine their efficacy in the inhibition of COX-2 activity and expression. Overall, compound A2 was the only one with a detectable inhibitory potential of COX-2 activity whilst two of its ether derivatives demonstrated improved ability in the inhibition of COX-2 expression.

Measurement of residual chemical shift anisotropies in compressed polymethylmethacrylate gels. Automatic compensation of gel isotropic shift contribution.

Mechanical compression of polymer gels provides a simple way for the measurement of residual chemical shift anisotropies, which then can be employed, on its own, or in combination with residual dipolar couplings, for structural elucidation purposes. Residual chemical shift anisotropies measured using compression devices needed a posteriori correction to account for the increase of the polymer to solvent ratio inside the swollen gel. This correction has been cast before in terms of a single-free parameter which, as shown here, can be simultaneously optimized along with the components of the alignment tensor while still retaining discriminating power of the different relative configurations as illustrated in the stereochemical analysis of α-santonin and 10-epi-8-deoxycumambrin B.

Complexation of sesquiterpene lactones with cyclodextrins: synthesis and effects on their activities on parasitic weeds.

Allelochemicals are safer, more selective and more active alternatives than synthetic agrochemicals for weed control. However, the low solubility of these compounds in aqueous media limits their use as agrochemicals. Herein, we propose the application of α-, ß- and γ-cyclodextrins to improve the physicochemical properties and biological activities of three sesquiterpene lactones: dehydrocostuslactone, costunolide and (-)-α-santonin. Complexation was achieved by kneading and coprecipitation methods. Aqueous solubility was increased in the range 100-4600% and the solubility-phase diagrams suggested that complex formation had been successful. The results of the PM3 semiempirical calculations were consistent with the experimental results. The activities on etiolated wheat coleoptiles, Standard Target Species and parasitic weeds were improved. Cyclodextrins preserved or enhanced the activity of the three sesquiterpene lactones. Free cyclodextrins did not show significant activity and therefore the enhancement in activity was due to complexation. These results are promising for applications in agrochemical design.

Discovery of Potent Small-Molecule Inhibitors of Ubiquitin-Conjugating Enzyme UbcH5c from α-Santonin Derivatives.

As a therapeutic target for antitumor necrosis factor (TNF)-α interventions, UbcH5c is one of the key ubiquitin-conjugating enzymes catalyzing ubiquitination during TNF-α-triggered nuclear factor kappa B (NF-κB) activation. In the present study, three series of analogues were designed and synthesized from α-santonin, and their UbcH5c inhibitory activities were screened by Western blotting and NF-κB luciferase assay. Further BIAcore, in-gel fluorescence imaging, and immunoprecipitation assays demonstrated that compound 6d exhibited robust and specific inhibition of UbcH5c, exceeding that of the positive compound 1 (IJ-5). Mechanistic investigations revealed that compound 6d preferentially bound to and inactivated UbcH5c by forming a covalent adduct with its active site Cys85. Furthermore, compound 6d exhibited potent anti-inflammatory activity against complete Freund's adjuvant-induced adjuvant arthritis in vivo. These findings suggest that the novel α-santonin-derived UbcH5c inhibitor 6d is a promising lead compound for the development of new antirheumatoid arthritis (RA) agent.

Quantification of santonin in eight species of Artemisia from Kazakhstan by means of HPLC-UV: Method development and validation.

Santonin, a powerful anthelmintic drug that was formely used to treat worms, is Artemisia cina's main constituent. However, due to its toxicity to humans, it is no longer in use. Kazakhstan is looking to introduce this plant as an anthelmintic drug for veterinary purposes, despite the known toxic properties of the santonin. The objective of this study was to develop a fast and specific method for the identification of santonin and its precise quantitation using HPLC-UV in order to avoid unnecessary intoxication, which is paramount for the development of veterinary medicines. The results obtained showed that santonin appears at around 5.7 minutes in this very reliable HPLC method. The validation of the method was performed by the investigation of parameters such as precision, accuracy, reproducibility and recovery. The method was used to identify and quantify santonin in leaves of A. scoparia, A. foetida, A. gmelinni, A. schrenkiana, A. frigida, A. sublesingiana, A terra-albae, and A. absinthium from Kazakhstan as well as in three different extracts of leaves of A. cina. This study has provided a faster and simpler method for the identification and quantification of this compound in other species of Artemisia of economic importance.

Synthesis of α-santonin derivatives for diminutive effect on T and B-cell proliferation and their structure activity relationships.

A new library of 20 compounds from α-santonin was synthesized and tested against Con-A induced T-cell proliferation and LPS-induced B-cell proliferation via MTT assay. The study resulted in the identification of potent immunosuppressant molecules, which were further screened along with α-santonin for Tumor Necrosis Factor Alpha (TNF-α) inhibitory activity. One of the molecules (7) at 10 µM showed equipotency to that of dexamethasone (1 µM conc.) used as a standard. Structure activity relationships of the synthesized compounds along with our earlier reported α-santonin derivatives have been studied. Inferences from the modifications carried out at all the three sites of α-santonin have been elaborated. Computational study of the active compounds shows TNF-α protein as its preferable target rather than Inosine Monophosphate Dehydrogenase (IMPDH).

Synthesis of α-santonin derived acetyl santonous acid triazole derivatives and their bioevaluation for T and B-cell proliferation.

A new series of α-santonin derived acetyl santonous acid 1,2,3-triazole derivatives were synthesised using Huisgen 1,3-dipolar cyclo-addition reaction (click chemistry approach) and evaluated for their in vitro inhibition activity on concanavalin A (ConA) induced T cell proliferation and lipopolysaccharide (LPS) induced B cell proliferation. Among the synthesised series, compounds 2-10 and 19 exhibited significant inhibition against ConA and LPS stimulated T-cell and B-cell proliferation in a dose dependent manner. More significantly compounds 4, 9-10 and 19 exhibited potent inhibition activity with remarkably lower cytotoxicity on the mitogen-induced T cell and B cell proliferation at 1 µM concentration. The compound 6 displayed potent immunosuppressive effects with ∼89% against LPS induced B-cell and ∼83% against ConA stimulated T-cell proliferation at 100 µM concentration without cytotoxicity. Compound 10 was more selective against B cell proliferation and exhibited 81% and 69% suppression at 100 and 1 µM concentration respectively. The present study led to the identification of several santonin analogs with reduced cytotoxicity and strong inhibition activity against the cell proliferation induced by the mitogens.

Novel platensimycin derivatives with herbicidal activity.

BACKGROUND: Faced with the need to develop herbicides with different modes of action on account of weed resistance to existing herbicides, the sesquiterpene lactones can be the starting point in the search for new bioactive compounds. Lumisantonin and five novel amides have been evaluated against two monocotyledons and three dicotyledons. RESULTS: An efficient and versatile synthesis of lumisantonin and the five novel amides has been accomplished from readily available α-santonin. These compounds were subjected to evaluation for their biological activity against Sorghum bicolor (sorghum), Allium cepa (onion), Cucumis sativus (cucumber), Solanum lycopersicum (tomato) and Bidens pilosa (beggartick). Lumisantonin has inhibited the development of the aerial parts of sorghum and onion by 76 and 67% at 1000 µM respectively. One of the novel amides has prevented the growth of shoots and radicles of sorghum by 80 and 71% at 1000 µM respectively. CONCLUSION: All of the tested compounds have been found to exhibit promising seed germination inhibition. We can conclude that lumisantonin was on average the most lethal against all plant species evaluated; however, two of the novel amides have exhibited inhibition selectivity against monocotyledons when compared with dicotyledons. © 2015 Society of Chemical Industry.

[Gastrostomy in dogs with magnetic compression technique combined with endoscopy].

OBJECTIVE: To evaluate the feasibility of endoscopic gastrostomy combined with magnetic compression techniques in dogs. METHODS: The magnetic compression device consisted of a couple of cylindrical magnets with a hole in the center. A 14-French gastric tube was inserted through the hole and fixed on the parent magnets. Four male mongrel dogs were selected randomly for the operation. The daughter magnet was placed into the dog's gastric body by a Zebra Guidewire under endoscopy, then a skin incision was performed on the left upper abdomen, through which the subcutaneous tissues and muscle were isolated up to the peritoneum. The parent magnet was placed on the surface of the peritoneum, which attracted the daughter magnet inside the gastric cavity automatically to compress the gastric wall and peritoneum. A needle was inserted through the gastric tube and punctured the tissue in the hole of the magnets repeatedly to form a fistula. After removing the needle, the gastrostomy was complete. The fistula was observed and its specimen was taken two weeks later under laparotomy. RESULTS: All the four operations were successfully performed, and the operation time was 11-15 minutes. The stoma was matured immediately, through which early enteral nutrition support was administered. The gastric wall and peritoneum were observed with well healing and no adhesion in the abdominal cavity under laparotomy two weeks after the operation. CONCLUSION: The gastrostomy performed by magnetic compression technique combined with endoscopy is convenient, minimally invasive and safe, which may be used in future clinical practice.

Design, synthesis and anticancer activity of Michael-type thiol adducts of α-santonin analogue with exocyclic methylene.

A series of Michael-type analogues were generated on the C-ring of α-santonin (α-methylene-γ-butyrolactone) upon reaction with various thiols. All the thiol adducts synthesized were evaluated for their anticancer activity against four human cancer cell lines (PC-3, HCT-15, A-549 and MCF-7). Bioassay results indicated that even though most of the synthesized compounds exhibited a good anticancer activity against various cancer cells in vitro, some of the compounds like 9e, 9g and 9q were found to be the most promising analogues in this series, with compound 9e showing IC50 values of 1.5 µM, 0.6 µM, 2.4 µM and 1.2 µM on PC-3, MCF-7, A-549 and HCT-116 cell lines respectively. Further, flow cytometry studies showed that MCF-7 cells treated with the compounds 9e, 9g and 9q were arrested in the sub G1 phase of the cell cycle in a concentration dependent manner. These lead molecules were further studied for NF-κB, p65 transcription factor inhibitory activity which confirmed concentration dependent inhibition against NF-κB, p65 with analogue 9e showing 57% inhibition at 2 µM, 9g showing 62% inhibition at 3 µM and 9q showing 54% inhibition at 2 µM concentration.