Anti-angiogenic effects of aqueous extract from Agrostemma githago L. seed in human umbilical vein endothelial cells via regulating Notch/VEGF, MMP2/9, ANG2, and VEGFR2.

Abnormal angiogenesis contributes to the pathogenesis of various diseases. The medicinal usage of Agrostemma githago L. seed (A. githago herein) has been stated in traditional medicine. This study aims to investigate the anti-angiogenic potential of aqueous extract of A. githago. In order to test the effect of A. githago extract, its impact on HUVECs, T98G, and HGF2PI2 cells was assessed by looking at cellular viability, changes in the distribution of cells in different phases of the cell cycle, induction of oxidative stress, and apoptosis. In addition, the release of VEGF, ANG2, and MMP2/9 factors, along with the expressions of the critical Notch signaling pathway players and VEGF receptors (VEGFR), was measured. Furthermore, a γ-secretase inhibitor (LY411575) was applied to determine whether Notch inhibition restores A. githago effects. As a further characterization, total phenolic and flavonoid contents of A. githago were estimated, and five triterpene saponin compounds were identified using LC-ESI-MS. In response to A. githago extract, a reduction in total cell viability, along with the induction of ROS and apoptosis, was detected. Exposure to the A. githago extract could modulate the release of VEGF and ANG2 from T98G and HUVECs, respectively. In addition, A. githago reduced the release of MMP2/9. Furthermore, Notch1, DLL4, and HEY2 transcripts and protein expressions were up-regulated, while VEGFR2 was down-regulated in treated HUVEC cells. Treatment with the A. githago extract resulted in a dose-dependent inhibition of AKT phosphorylation. Inhibition of Notch signaling retrieved the viability loss, reduced intracellular ROS, and alleviated the impaired tube formation in A. githago-treated HUVECs. Overall, these data underscore the anti-angiogenic potential of A. githago via inducing apoptosis, modifying the expression levels of VEGF/VEGFR2, and impacting the release of MMP2/9 and ANG2, effects that are most probably modulated through the Notch/VEGF signaling axis.

An unusual type I ribosome-inactivating protein from Agrostemma githago L.

Agrostemma githago L. (corn cockle) is an herbaceous plant mainly growing in Europe. The seeds of the corn cockle are toxic and poisonings were widespread in the past by consuming contaminated flour. The toxic principle of Agrostemma seeds was attributed to triterpenoid secondary metabolites. Indeed, this is in part true. However Agrostemma githago L. is also a producer of ribosome-inactivating proteins (RIPs). RIPs are N-glycosylases that inactivate the ribosomal RNA, a process leading to an irreversible inhibition of protein synthesis and subsequent cell death. A widely known RIP is ricin from Ricinus communis L., which was used as a bioweapon in the past. In this study we isolated agrostin, a 27 kDa RIP from the seeds of Agrostemma githago L., and determined its full sequence. The toxicity of native agrostin was investigated by impedance-based live cell imaging. By RNAseq we identified 7 additional RIPs (agrostins) in the transcriptome of the corn cockle. Agrostin was recombinantly expressed in E. coli and characterized by MALDI-TOF-MS and adenine releasing assay. This study provides for the first time a comprehensive analysis of ribosome-inactivating proteins in the corn cockle and complements the current knowledge about the toxic principles of the plant.

A new acetylated triterpene saponin from Agrostemma githago L. modulates gene delivery efficiently and shows a high cellular tolerance.

Transfection is the process to deliver nucleic acid into eukaryotic cells. Different transfection techniques already exist. However, they can be expensive and toxic toward subjected cells. Previous research shed light on natural occurring molecules called triterpene saponins that have great potential for the non-viral gene delivery. Using a combination of different chromatographic techniques and in vitro transfection bioassays, a new triterpenoid saponin (agrostemmoside E) from the plant Agrostemma githago L. was isolated. Agrostemmoside E was characterized by mass spectrometry, intense NMR spectroscopy and was identified as 3-{O-ß-D-Galactopyranosyl-(1→2)]-[ß-D-xylopyranosyl-(1→3)]-ß-D-glucuronopyranosyl} quillaic acid 28-O-{[ß-D-4,6-di-(O-acetyl)-glucopyranosyl-(1→3)]-[ß-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)}-[3,4-di-(O-acetyl)-ß-D-quinovopyranosyl-(1→4)]-ß-D-fucopyranoside ester. Agrostemmoside E has a great potential for delivery of gene loaded nanoplexes and increased the transfection efficiency by 70% compared to 2% without agrostemmoside E. By comparative toxicity studies, we show that agrostemmoside E can be applied at high concentrations without toxicity, justifying its use as a new tool for gene transfections.

Aqueous extract of Agrostemma githago seed inhibits caspase-3 and induces cell-cycle arrest at G1 phase in AGS cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: A weed plant belonging to the Caryophyllaceae family, Agrostemma githago is used in folk medicine to treat cancers and warts. AIM OF THE STUDY: The aim of this study was to evaluate the cytotoxic effect of the aqueous extract of A. githago seed on gastric cancer cell line (AGS) and to investigate the mechanism of apoptosis induction in these cells. MATERIALS AND METHODS: Seeds of A. githago were collected from the suburban area of Ardabil Province, northwest Iran. After preparing the aqueous extract, dry matter was harvested with the lyophilizing technique. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxicity. Apoptotic cells were detected by staining with ethidium bromide/acridine orange (EB/AO). Diamidino-2-phenylindole (DAPI) staining was used for cell-cycle analysis with a flow cytometer. The annexin V binding level, caspase-3 activity, and B-cell lymphoma-2 (Bcl-2) level were also measured to confirm apoptotic cell death. RESULTS: After the aqueous extract of A. githago seed was incubated for 24, 48, and 72 h, inhibited cell growth was observed with IC50 values of 13.51 ± 0.7, 4.37 ± 1.01, and 2.42 ± 0.8 µg/ml, respectively. The EB/AO staining method demonstrated that the extract exerts its cytotoxic effect mainly via apoptosis, in accordance with the annexin V, blc-2, and caspase-3 results. The extract showed a concentration-dependent increase in annexin V binding to externally exposed phosphatidylserine as well as caspase-3 activity. The bcl-2 protein level showed a proportionate decrease with the increase in extract concentration. The cell-cycle analysis revealed that the extract can arrest cells at the G1 checkpoint. CONCLUSION: The findings of this study revealed the cytotoxic effect of the aqueous extract of A. githago seed on gastric cancer cells (AGS) mainly via apoptosis and the cell-cycle arrest at the G1 checkpoint. Therefore, the extract can be potentially used in gastric cancer therapy in vitro.

Environmental conditions during early life determine the consequences of inbreeding in Agrostemma githago (Caryophyllaceae).

In an inbred population, selection may reduce the frequency of deleterious recessive alleles through a process known as purging. Empirical studies suggest, however, that the efficacy of purging in natural populations is highly variable. This variation may be due, in part, to variation in the expression of inbreeding depression available for selection to act on. This experiment investigates the roles of life stage and early-life environment in determining the expression of inbreeding depression in Agrostemma githago. Four population-level crosses ('self', 'within', 'near' and 'far') were conducted on 20 maternal plants from a focal population. Siblings were planted into one of three early environmental treatments with varying stress levels. Within the focal population, evidence for purging of deleterious recessive alleles, as well as for variation in the expression of inbreeding depression across the life cycle was examined. In addition, the effect of early environment on the expression of inbreeding depression and the interaction with cross-type was measured. We find that deleterious recessive alleles have not been effectively purged from our focal population, the expression of inbreeding depression decreases over the course of the life cycle, and a stressful early environment reduces the variance in inbreeding depression expressed later in life, but does not consistently influence the relative fitness of inbred versus outcrossed individuals.

Triterpene glycosides from Agrostemma gracilis.

Four triterpene saponins, agrostemmosides A-D were isolated from the methanol extract of Agrostemma gracilis. The structures of the compounds were determined as 3-O-beta-D-xylopyranosyloleanolic acid 28-O-beta-D-glucopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->6)]-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyloleanolic acid 28-O-beta-D-glucopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->6)]-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3-O-beta-D-xylopyranosylechinocystic acid 28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester, 3-O-beta-D-xylopyranosylechinocystic acid 28-O-beta-D-glucopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->6)]-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester by a combination of one- and two-dimensional NMR techniques, and mass spectrometry. To the best of our knowledge this is the first phytochemical report on A. gracilis, and echinocystic acid saponins were encountered for the first time in Caryophyllaceae family.

Synthesis, resolution, and VCD analysis of an enantiopure diazaoxatricornan derivative.

Using simple organic synthetic transformations, a novel diazaoxatricornan derivative, the 12 c-methyl-12-phenyl-8-propyl-12,12 c-dihydro-8 H-4-oxa-8,12-diazadibenzo[ cd, mn]pyrene ( 6a), was prepared. This novel chiral cup-shaped molecule was isolated in racemic form and in excellent yield after the addition of methyl lithium to the BF 4 salt of a novel unsymmetrical diazaoxatriangulenium cation. Compound 6a was found to be stable under classical laboratory conditions-something not obvious considering the extreme stability of the carbenium ion precursor, the electron-rich nature of the core, and the strain induced by the pyramidalization of the central carbon. The enantiomers were readily separated by chiral stationary phase chromatography, and the absolute configuration of (-)-( S)- 6a was determined by a comparison of the experimental and theoretical vibrational circular dichroism (VCD) spectra. This isolation of (-)-( S)- 6a and (+)-( R)- 6a constitutes thus the first report of a nonracemic closed-capped chiral bowl molecule for which the chirality is due to the intrinsic dissymmetry of the central core of the structure only.

Antihypercholesterolaemic and antioxidant activity assessment of some plants used as remedy in Turkish folk medicine.

Ethanolic and aqueous extracts from five plant species used in Turkish traditional medicine were evaluated for in vivo hypercholesterolaemic and antioxidant activities: Agrostemma githago L., Potentilla reptans L., Thymbra spicata var. spicata L., Urtica dioica L. and Viscum album var. album L. We assayed the effects of the administration of plant extracts on serum total cholesterol, triglyceride, HDL-C, LDL-C, glucose, AST and ALT concentrations in mice fed with cholesterol-rich diet. In addition, plasma TAA, MDA and NO(x) levels in the same animals were assayed. All the aqueous plant extracts did not affect the serum cholesterol concentration. However, the ethanolic extracts of Agrostemma githago, Thymbra spicata and Viscum album decreased the serum cholesterol concentration in the mice fed with high-cholesterol diet without inducing any gastric damage. The ethanolic extracts of Thymbra spicata, Viscum album, Potentilla reptans and Urtica dioica and the aqueous extract of Agrostemma githago increased the serum HDL concentration, whereas the ethanolic extracts of Agrostemma githago, Thymbra spicata, Viscum album and Urtica dioica decreased the serum LDL-C concentration. Thymbra spicata and Viscum album were observed to decrease the serum triglyceride concentration. Among the plant extracts studied, the ethanolic extracts of Thymbra spicata significantly decreased the MDA level in mice. The ethanolic extract of Potentilla reptans increased in NO(x). None of these plants showed statistically prominent activity on plasma TAA. Results of the present study indicated that the ethanolic extracts of Agrostemma githago, Thymbra spicata and Viscum album showed potent hypocholesterolaemic activity in the mice fed with a diet containing high-cholesterol.

Enhancement of cytotoxicity of lectins by Saponinum album.

Saponinum album, a mixture of saponins with an aldehyde function bound at C4 from Gypsophila species, increased the cytotoxicity of lectins like agrostin and saporin by enhancing its penetration through the cell membrane. The effect was attenuated by latrunculin, an inhibitor of endocytosis, and by bafilomycin, an inhibitor of transport from early to late endosomes and lysosomes. In contrast, the effect was not influenced by brefeldin A that causes dissolution of the Golgi stacks and by the addition of different monosaccharides. The toxicity of other peptidic toxins was only slightly enhanced by the saponins indicating a specific interaction between lectins and saponins present in Caryophyllaceae.

Metabolism of the nonylphenol isomer [ring-U-14C]-4-(3',5'-dimethyl-3'-heptyl)-phenol by cell suspension cultures of Agrostemma githago and soybean.

The biotransformation of the nonylphenol isomer [ring-U-14C]-4-(3',5'-dimethyl-3'-heptyl)-phenol (4-353-NP, consisting of two diastereomers) was studied in soybean and Agrostemma githago cell suspension cultures. With the A. githago cells, a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v) was used, in order to produce higher concentrations and amounts of 4-353-NP metabolites for their identification; 4-353-NP was applied via the n-hexadecane phase. Initial concentrations of [14C]-4-353-NP were 1 mg L(-1) (soybean), and 5 and 10 mg L(-1) (A. githago). After 2 (soybean) and 7 days (A. githago) of incubation, the applied 4-353-NP was transformed almost completely by both plant species to four types of products: glycosides of parent 4-353-NP, glycosides of primary 4-353-NP metabolites, nonextractable residues and unknown, possibly polymeric materials detected in the media. The latter two products emerged especially in soybean cultures. Portions of primary metabolites amounted to 19-22% (soybean) and 21-42% of applied 14C (A. githago). After liberation from their glycosides, the primary 4-353-NP metabolites formed by A. githago were isolated by HPLC and examined by GC-EIMS as trimethylsilyl derivatives. In the chromatograms, eight peaks were detected which due to their mass spectra, could be traced back to 4-353-NP. Seven of the compounds were side-chain monohydroxylated 4-353-NP metabolites, while the remaining was a (side-chain) carboxylic acid derivative. Unequivocal identification of the sites of hydroxylation/oxidation of all transformation products was not possible. The main primary metabolites produced by A. githago were supposed to be four diastereomers of 6'-hydroxy-4-353-NP (about 80% of all products identified). It was concluded that plants contribute to the environmental degradation of the xenoestrogen nonylphenol; the toxicological properties of side-chain hydroxylated nonylphenols remain to be examined.

Cytotoxic activity of the seeds from Agrostemma githago var. githago.

In our investigations aqueous extracts of the seeds from Agrostemma githago L. proved to be remarkably more cytotoxic in comparison with isolated agrostemmasaponins in equal concentrations. A combination of agrostemmasaponin 1 with a formyl function attached to triterpene position 4 together with agrostin, a ribosome-inactivating protein (RIP type 1; M (r): 27 kDa) which we isolated from the seed material, resulted in a markedly increased cytotoxicity. In analogy to the well-known lectin-like mechanism of action which can be inhibited by different monosaccharides, we tested seven different sugars known for their affinity to prevent cell recognition and thereby detoxify lectins. None of the tested monosaccharides diminished toxicity values significantly. This suggests that agrostin, combined with agrostemmasaponin 1, has a different mode of action to penetrate through the cell membrane than lectins. On the basis of these facts we concluded that both compounds work as a functional unity showing a mechanism of "cooperative toxicity".