An efficient method for the preparative isolation and purification of alkaloids from Gelsemium by using high speed counter-current chromatography and preparative HPLC.

We established an efficient method using high-speed countercurrent chromatography (HSCCC) combined with preparative high-performance liquid chromatography (prep-HPLC) for isolating and purifying Gelsemium elegans (G. elegans) alkaloids. First, the two-phase solvent system composed of 1% triethylamine aqueous solution/n-hexane/ethyl acetate/ethanol (volume ratio 4:2:3:2) was employed to separate the crude extract (350 mg) using HSCCC. Subsequently, the mixture that resulted from HSCCC was further separated by Prep-HPLC, resulting in seven pure compounds including: 14-hydroxygelsenicine (1, 12.1 mg), sempervirine (2, 20.8 mg), 19-(R)-hydroxydihydrogelelsevirine (3, 10.1 mg), koumine (4, 50.5 mg), gelsemine (5, 32.2 mg), gelselvirine (6, 50.5 mg), and 11-hydroxyhumanmantenine (7, 12.5 mg). The purity of these seven compounds were 97.4, 98.9, 98.5, 99, 99.5, 96.8, and 85.5%, as determined by HPLC. The chemical structures of the seven compounds were analyzed and confirmed by electrospray ionization mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H NMR), and 13 C-nuclear magnetic resonance (13 C NMR) spectra. The results indicate that the HSCCC-prep-HPLC method can effectively separate the major alkaloids from the purified G. elegans, holding promising prospects for potential applications in the separation and identification of other traditional Chinese medicines.

Counterion-Controlled Formation of Layered Honeycomb and Polythreading Uranyl Networks and the Highly Sensitive and Selective Detection of Fe3+ in Aqueous Media.

Under hydrothermal conditions, six uranium coordination polymers were obtained by employing the ligand of tris(2-carboxyethyl) isocyanurate (H3tci) and different combinations of d-block metal ions (Mn2+, Co2+, Zn2+, Ni2+) or N-donors (triethylamine (Et3N), 2,2'-bipyridine (2,2'-bipy)). Three uranium polymers [(UO2)2(tci)2]1/2-·[Mn2(µ2-O)(H2O)8]1/2+·2H2O (1), [(UO2)2(tci)2]1/2-·[Co2(µ2-O)(H2O)8]1/2+ (2), and [(UO2)2(tci)2]1/2-·[Zn2(µ2-O)(H2O)8]1/2+·2H2O (3) containing transition metal hydrated ions, crystallized as two-dimensional coordination polymers with the common (6, 3) net topology. X-ray crystal structures of 1-3 display that they have similar honeycomb-like frameworks with all ligands bis-chelating. [UO2(tci)]-3·[Ni(H2O)6]2+(H3O)+·2H2O (4) and [UO2(tci)]-·[NH(CH2CH3)3]1/3+·(2H3O+)1/3 (5) are made up of four interlocked sets and exhibit the 4-fold-interpenetrated frameworks. [UO2(tci)]-·(C10H9N2)+·H2O (6) comprises the 2,2'-bipy cation as counterion and represents a 2D grid layered structure. Additional metals and the N-donors are all free in the complexes, acting as the templates and compensation of the charge equilibrium. The solid-state emission spectra indicate that all of the synthesized compounds own fluorescence emissions. Furthermore, the results of the quenching ability of Fe3+ for complexes 5 and 6 exhibit their highly sensitive and selective detection for Fe3+ ions. Moreover, the uranium complexes can be used as a potential probe for Fe3+ in aqueous solutions.

In vivo confocal microscopy of pre-Descemet corneal dystrophy associated with X-linked ichthyosis: a case report.

BACKGROUND: Pre-Descemet corneal dystrophy (PDCD) is characterized by the presence of numerous, tiny, polymorphic opacities immediately anterior to Descemet membrane, which is a rare form of corneal stromal dystrophy and hard to be diagnosed. In vivo confocal microscopy (IVCM) is a useful tool to examine the minimal lesions of the cornea at the cellular level. In this article, we report a rare case of PDCD associated with X-linked ichthyosis and evaluate IVCM findings. CASE PRESENTATION: We present a 34-year-old male Chinese patient with PDCD associated with X-linked ichthyosis. Slit-lamp biomicroscopy showed the presence of tiny and pleomorphic opacities in the posterior stroma immediately anterior to Descemet membrane bilaterally. IVCM revealed regular distributed hyperreflective particles inside the enlarged and activated keratocytes in the posterior stroma. Hyperreflective particles were also observed dispersedly outside the keratocytes in the anterior stroma. Dermatological examination revealed that the skin over the patient's entire body was dry and coarse, with thickening and scaling of the skin in the extensor side of the extremities. PCR results demonstrated that all ten exons and part flanking sequences of STS gene failed to produce any amplicons in the patient. CONCLUSIONS: IVCM is useful for analyzing the living corneal structural changes in rare corneal dystrophies. We first reported the IVCM characteristics of PDCD associated with X-linked ichthyosis, which was caused by a deletion of the steroid sulfatase (STS) gene, confirmed by gene analysis.

Central retinal artery occlusion associated with persistent truncus arteriosus and single atrium: a case report.

BACKGROUND: Central retinal artery occlusion (CRAO) is an ocular emergency and most of the cases present with painless sudden persistent loss of vision in the range of counting fingers to perception of light. The presentation of CRAO is associated with a variety of medical conditions. We report a rare case of CRAO associated with persistent truncus arteriosus (PTA) and single atrium in a female patient. CASE PRESENTATION: A 23-year-old woman was admitted due to sudden painless visual loss in the left eye. On examination visual acuity of light-perception was noted in the left eye with a left relative afferent pupillary defect. Fundoscopic examination revealed retinal ischemic whitening, constriction of the arteriole and venule with segmentation and typical "cherry-red spot" suggesting CRAO. The patient was treated with ocular massage and anterior chamber paracentesis. She was commenced on 150 mg of aspirin and also received hyperbaric oxygen therapy. An echocardiogram revealed PTA and single atrium. A diagnosis of CRAO associated with PTA and single atrium was made. CONCLUSION: The ophthalmologist should enquire about congenital and acquired cardiac abnormalities in patients with CRAO and consider such abnormalities to be possible sources of emboli.

Effect of 0.02% and 0.01% atropine on ocular biometrics: A two-year clinical trial.

Background: Several studies have shown that various concentrations of low-concentration atropine can reduce myopia progression and control axial elongation safely and efficiently in children. The aim of this study was to evaluate the effects of 0.02% and 0.01% atropine on ocular biometrics. Methods: Cohort study. 138 and 142 children were randomized to use either 0.02% or 0.01% atropine eye drops, respectively. They wore single-vision (SV) spectacles, with one drop of atropine applied to both eyes nightly. Controls (N = 120) wore only SV spectacles. Ocular and corneal astigmatism were calculated using Thibos vector analysis and split into J0 and J45. Results: The changes in cycloplegic spherical equivalent refraction (SER) and axial length (AL) were -0.81 ± 0.52D, -0.94 ± 0.59D, and -1.33 ± 0.72D; and 0.62 ± 0.29 mm, 0.72 ± 0.31 mm, and 0.89 ± 0.35 mm in the 0.02% and 0.01% atropine and control groups, respectively (all P < 0.05). Both anterior chamber depth (ACD) and ocular astigmatism (including J0) increased, and lens power decreased in the three groups (all P < 0.05). However, there were no differences in the changes in ACD, ocular astigmatism, and lens power among the three groups (all P > 0.05). Intraocular pressure (IOP), corneal curvature, ocular astigmatism J45, and corneal astigmatism (including J0 and J45) remained stable over time in the three groups (all P > 0.05). The contributions to SER progression from the changes in AL, lens and corneal power of the three groups were similar (P > 0.05). The contribution of AL change alone to the change in SER was 56.3%, 63.4% and 78.2% in the above corresponding three groups. Conclusions: After 2 years, 0.02% and 0.01% atropine had no clinical effects on corneal and lens power, ocular and corneal astigmatism, ACD or IOP compared to the control group. 0.02% and 0.01% atropine helped to control myopia progression mainly by reducing AL elongation.

Protocol to characterize target components of Gelsemium using an in-house herb database and high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Currently, the identification of herb metabolites is challenging due to a lack of clear standards. Here, using Gelsemium as an example, we present a protocol for characterizing target components of herbs. This approach utilizes high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry guided by an in-house herb metabolite database based on reported studies and mass spectrometry. We describe steps for creating an in-house database, preparing and detecting samples, processing data, and characterizing compounds. This approach offers a reference for future research on the identification of herb metabolites. For complete details on the use and execution of this protocol, please refer to Liu et al. (2017).1.

Comparison of 0.02% atropine eye drops, peripheral myopia defocus design spectacle lenses, and orthokeratology for myopia control.

CLINICAL RELEVANCE: There are many methods to control the progression of myopia. However, it is currently unknown which method could better control myopia progression: 0.02% atropine eye drops, peripheral myopic defocus design spectacle lenses (PMDSL), or orthokeratology (OK). BACKGROUND: To compare the efficacy of 0.02% atropine, PMDSL, and OK to control axial length (AL) elongation in children with myopia. METHODS: This study was analysed based on a previous cohort study (0.02% atropine group) and retrospective data (PMDSL and OK group). Overall, 387 children aged 6-14 years with myopia - 1.00D to - 6.00D in the three groups were divided into four subgroups according to age and spherical equivalent refraction (SER). The primary outcome was changed in AL over 1-year. RESULTS: The mean axial elongation was 0.30 ± 0.21 mm, 0.23 ± 0.16 mm, and 0.17 ± 0.19 mm in the 0.02% atropine, PMDSL, and OK groups, respectively. Multivariate linear regression analyses showed significant differences in axial elongation among the three groups, especially in children aged 6-10, but not in children aged 10.1-14; the corresponding axial elongation was 0.35 ± 0.21 mm, 0.23 ± 0.17 mm, and 0.21 ± 0.20 mm (P < 0.05 between any two groups, except between PMDSL and OK groups at P > 0.05) and 0.22 ± 0.20 mm, 0.21 ± 0.13 mm, and 0.13 ± 0.18 mm (P < 0.05 between any two groups, except between 0.02% atropine and PMDSL groups at P > 0.05) in children with SER from - 1.00D to - 3.00D and from - 3.01D to - 6.00D, respectively. CONCLUSIONS: Within the limits of this study design and using only the current brand of PMDSL, OK appeared to be the best method, followed by PMDSL and then 0.02% atropine, for controlling AL elongation over one year. However, different effects were found in the various age and SER subgroups.

[Effects of pressure bionic culture on the morphology of corneal endothelium cells].

OBJECTIVE: To investigate the effect of pressure bionic culture on the morphology and function of rabbit corneal endothelial cells. METHODS: Corneal endothelial cells were separated and purified by tearing apart the descemet and digesting with trypsin and EDTA, then cultured in the plate. The cells were divided into two groups: group A were cultured under atmosphere; cells exposed to 2 kPa (14.66 mm Hg) pressure in vitro was group B; the morphology and growth pattern of cells were observed by inverted microscope; cells origin were identified by neuron-specific enolase immunoassay. Cellular changes in the structure were observed by HE staining and scanning and transmission electron microscopy (SEM and TEM) analysis. Cells activity was detected by flow cytometry. RESULTS: NSE antibody of the primary corneal endothelial cells was positive without corneal epithelial cells and corneal stroma cells. Two groups of cells were cultured for 120-144 h respectively, the morphology was flat, polygon, most of cells were hexagon and abundant cytoplasms in group B (pressure bionic culture), but in group A, the cells size was not uniform and there were much granules in the cytoplasm. There was no difference in the time of formation of monolayer in two groups. SEM showed that cells exposed to pressure connected tightly and the surface was rich in microvilli, extended foot processes and attached to the substrate tightly, while cells cultured under atmosphere with more off-chip. In group B, Annexin-FITC/PI detection of apoptosis showed cell survival rate was 98.2%, early apoptosis rate was 0.7%, late apoptosis rate was 1.0%, death rate was 0.1%; the corresponding data were 92.2%, 5.2%, 2.3%, and 0.3% in group A, respectively; There was statistically significant difference between the two groups (χ(2) = 594.0, P < 0.01). After cultured for 96 h, the expression of ZO-1 protein in cells exposed to pressure was higher than those in control. CONCLUSIONS: The biological activity of endothelial cells is regulated positively by bionic pressure. The establishment of a new biomimetic pressure model will help to investigate the physiological function and injury repair of corneal endothelial cells in vitro.

Improved method for inducing chronic atrophic gastritis in mice.

BACKGROUND: Chronic atrophic gastritis (CAG) is a common disease of the digestive system with pathological characteristics of a decreasing number, or disappearance, of inherent glands of the gastric mucosa. CAG has been defined as a precancerous condition of gastric cancer. Intestinal metaplasia or intraepithelial neoplasia accompanying atrophied glands of the stomach is regarded as one of the most important precancerous lesions of gastric cancer. As a common malignant tumour, gastric cancer remains without a satisfactory therapy and its pathogenesis remains unclear, seriously threatening human life. Therefore, some scholars have proposed to prevent the incidence of gastric cancer by avoiding precancerous lesions. If CAG can be reversed, the incidence of gastric cancer can be substantially reduced. To reverse and prevent CAG and study its pathogenesis and therapy, it is necessary to develop an ideal, safe, stable, animal model. AIM: To study a rapid, stable, and safe method of establishing a mouse model of human CAG. METHODS: Six-week-old Kunming mice were divided into a phosphate buffered solution control group, a Helicobacter pylori (H. pylori) group, an N-methyl-N'-nitroguanidine (MNNG) group, an ammonia water group, and a group combining H. pylori, MNNG, and ammonia water (hereinafter referred to as the combined group). The mice were administrated with drinking water containing ammonia or infected with H. pylori through gavage. At the 30th, 60th, 90th, and 120th day after the last H. pylori infection, mice were selected randomly to collect their gastric mucosa for hematoxylin eosin staining, terminal nick-end labelling staining detection, and immunohistochemical staining for Bax and Bcl-2. In addition, H. pylori was isolated, cultured, and identified, and its extent of colonisation calculated. Blood was collected to detect inflammatory factors interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α and immune function markers CD4 and CD8 to confirm successful establishment of the CAG model. RESULTS: The combined group showed slight CAG at the 90th day and moderate CAG at the 120th day, while other groups did not show CAG at that time. CONCLUSION: The combination of H. pylori, MNNG, and ammonia is an effective method of developing a mouse model of human CAG.

4-Hydr-oxy-1-oxo-1,2-dihydro-phthalazine-6,7-dicarboxylic acid dihydrate.

In the crystal structure of the title compound, C(10)H(6)N(2)O(6)·2H(2)O, the OH and NH groups each serve as a hydrogen-bond donor to one acceptor site whereas the water mol-ecules each serve as a hydrogen-bond donor to two acceptor sites. The hydrogen-bonding scheme gives rise to a three-dimensional network.

Mechanism of combined use of vitamin D and puerarin in anti-hepatic fibrosis by regulating the Wnt/ß-catenin signalling pathway.

AIM: To reveal the protective mechanism of the combined use of vitamin D and puerarin in the progression of hepatic fibrosis induced by carbon tetrachloride (CCl4). METHODS: Eight-week-old male Wistar rats were randomly divided into a normal control group (C group), a CCl4 group (CCl4 group), a vitamin D group (V group), a puerarin group (P group), and a combined group of vitamin D and puerarin (V + P group), each of which contained ten rats. In this way, we built a rat model of CCl4-induced hepatic fibrosis with intervention by vitamin D, puerarin, or a combination of the two. After eight weeks, the mice were sacrificed to collect serum and liver specimens. Blood was collected to detect the hyaluronic acid (HA). We also measured hydroxyproline (Hyp) and prepared paraffin sections of liver. After Sirius red staining, the liver specimens were observed under a microscope. RT-PCR and western blot analysis were adopted to detect the mRNA and the protein levels of Collagen I, Collagen III, Wnt1, and ß-catenin in the liver tissues, respectively. RESULTS: Hepatic fibrosis was observed in the CCl4 group. In comparison, hepatic fibrosis was attenuated in the V, P, and V + P groups: the HA level in blood and the Hyp level in liver were reduced, and the mRNA levels of Collagen I, Collagen III, Wnt, and ß-catenin in liver were also decreased, as well as the protein levels of Wnt1 and ß-catenin. Among these groups, the V + P group demonstrated the greatest amelioration of hepatic fibrosis. CONCLUSION: The combined application of vitamin D and puerarin is capable of alleviating CCl4-induced hepatic fibrosis of rats. As to the mechanism, it is probably because the combined use is able to silence the Wnt1/ß-catenin pathway, suppress the activation of hepatic stellate cells, and reduce the secretion of collagen fibers, therefore improving the anti-hepatic fibrosis effect.

Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina.

AIM: To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells (EPCs) in a mouse model of laser-induced retinal injury. METHODS: EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes: 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), 1,1'-dilinoleyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein (DiI-AcLDL), and green fluorescent protein (GFP). The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo. RESULTS: EPCs labeled with CFSE and DiI-AcLDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and DiI-AcLDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina. CONCLUSION: The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and DiI-AcLDL are suitable for short-term EPC-labeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

Pseudomonas aeruginosa endophthalmitis caused by accidental iatrogenic ocular injury with a hypodermic needle.

Iatrogenic traumatic endophthalmitis is a rare but serious ocular infection that can lead to severe vision loss. A 44-year-old man presented with pain and decreased vision in the right eye 4 hours after injury with a hypodermic needle during irrigation of his eye. Slit-lamp examination revealed a penetrating corneal puncture and iris hole in the right eye. Twenty hours later, his visual acuity had decreased to hand motion, and severe fibrinoid uveitis was noted. He immediately underwent irrigation of the anterior chamber and intravitreal antibiotic injection. The right eye became painful again, and emergent vitrectomy combined with lensectomy was performed along with intravitreal antibiotic administration. The patient remained stable during the 2-month follow-up. Standard practice should be adopted when irrigating the eye to prevent this type of injury, and emergent surgical intervention is very important to preserve visual function.

Tracking of CFSE-labeled endothelial progenitor cells in laser-injured mouse retina.

BACKGROUND: Endothelial progenitor cells (EPCs) transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in laser-injured mouse retina. METHODS: EPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 µmol/L CFSE at 37°C was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells. RESULTS: EPCs labeled with 5 µmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks. CONCLUSION: EPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.