Separation of polysaccharides from Lycium barbarum L. by high-speed countercurrent chromatography with aqueous two-phase system.

The traditional method for isolation and purification of polysaccharides is time-consuming. It often involves toxic solvents that destroy the function and structure of the polysaccharides, thus limiting in-depth research on the essential active ingredient of Lycium barbarum L. Therefore, in this study, high-speed countercurrent chromatography (HSCCC) and aqueous two-phase system (ATPS) were combined for the separation of crude polysaccharides of Lycium barbarum L. (LBPs). Under the optimized HSCCC conditions of PEG1000-K2HPO4-KH2PO4-H2O (12:10:10:68, w/w), 1.0 g of LBPs-ILs was successfully divided into three fractions (126.0 mg of LBPs-ILs-1, 109.9 mg of LBPs-ILs-2, and 65.4 mg of LBPs-ILs-3). Moreover, ATPS was confirmed as an efficient alternative method of pigment removal for LBPs purification, with significantly better decolorization (97.1 %) than the traditional H2O2 method (88.5 %). Then, the different partitioning behavior of LBPs-ILs in the two-phase system of HSCCC was preliminarily explored, which may be related to the difference in monosaccharide composition of polysaccharides. LBPs-ILs-1 exhibited better hypoglycemic activities than LBPs-ILs-2 and LBPs-ILs-3 in vitro. Therefore, HSCCC, combined with aqueous two-phase system, was an efficient separation and purification method with great potential for separating and purifying active polysaccharides in biological samples.

Identification of anti-photoaging components of Olea europaea leaves based on spectrum-effect relationship.

In this study, to identify bioactive components of Olea europaea leaves extract (OLE), chemometrics analyses including bivariate correlation analysis and partial least squares regression were used to establish the relationships between the chromatograms and anti-photoaging effect of OLE samples. Firstly, the fingerprint of olive leaves extract was determined by high-performance liquid chromatography (HPLC). Photoaging models of HaCaT cells were established by UVB irradiation. The photoaging resistance of OLE was evaluated by cell viability using the MTT assay. Chemometrics analyses showed that compounds 14, 19, 20, 24, 26, and 28 might be the major anti-photoaging components of OLE. Furthermore, after separation by HSCCC and NMR identification, compound 19 is luteoloside and compound 24 is oleuropein. Oleuropein and luteoloside were docked with collagenase (MMP-1), stromelysin (MMP-3), and gelatinase (MMP-9), respectively. The results showed that oleuropein and luteoloside inhibited their activity by directly interacting with MMP-1, MMP-3, and MMP-9, thereby exhibiting anti-photoaging activity. The current bioassay and spectrum-effect relationships are proper for associating sample quality with the active ingredient, and our finding would provide foundation and further understanding of the quality evaluation and quality control of Olea europaea.

Macular pigment optical density responses to different levels of zeaxanthin in patients with high myopia.

PURPOSE: Measurement of macular pigment optical density (MPOD) can be conducted to assist in the diagnosis of multiple fundus diseases. METHODS: Fifty-four subjects with high myopia were prospectively recruited for a 3-month clinical trial. Detailed ophthalmologic examinations and MPOD measurements were performed. The subjects in each high myopia category group were randomly subdivided into 5 intervention groups, including a low-dose Lycium barbarum group (10 g Lycium barbarum, containing 10 mg of zeaxanthin and 1 mg of lutein), low-dose control group (1 mg of lutein), high-dose Lycium barbarum group (20 g of Lycium barbarum, containing 20 mg of zeaxanthin and 2 mg lutein), high-dose control group (2 mg of lutein), and a blank control group. Differences in the MPODs among the high myopia groups were calculated with Welch two-sample t tests. A linear mixed-effects model was constructed and Pearson's correlation analysis was performed to determine correlations between MPOD and other factors. The MPOD values at baseline and the 3-month follow-up were compared with the Mann-Whitney test. RESULTS: The category 1 group had a significantly higher MPOD than the category 2 (P = 0.02) and category 3 groups (P < 0.001). The category 2 group had a significantly higher MPOD than the category 3 group (P < 0.001). The MPOD significantly decreased with increasing axial length (AL) and decreasing best-corrected visual acuity (BCVA) in the category 1-3 groups and with increasing age and increasing intraocular pressure (IOP) in the category 2-3 groups. The MPOD was significantly higher in the group who received high-dose zeaxanthin from Lycium barbarum than in the group who received high-dose lutein at 3 months (P = 0.0403), while no significant difference was identified between the low-dose zeaxanthin group and low-dose lutein group (P = 0.1117). CONCLUSIONS: The MPOD was negatively correlated with the category of high myopia. Supplementation of zeaxanthin from Lycium barbarum is beneficial in preventing the loss of macular pigment of high myopia patients. TRIAL REGISTRATION: Trial registration number and date of registration: ChiCTR2100046748.

Preparation of highly purified oleuropein by combinative technology off line of HSCCC-PHPLC based on dual wavelength.

Oleuropein is the main active substance in foods or functional foods produced from olive (Olea europaea L.) leaves. In the present study, the combinative technology off line of HSCCC-PHPLC based on dual wavelength was used to separate highly purified oleuropein from oleuropein extract. Response surface methodology was used to optimize the conditions of HSCCC. Furthermore, a large amount of higher purified oleuropein was obtained through HSCCC at the wavelength of 254 nm, and oleuropein with the purity greater than 98.5% was obtained by PHPLC at the wavelength of 300 nm. Finally, the purity and structure identification of highly purified oleuropein were determined by various methods and its stability was investigated. As a result, oleuropein was stable in solution, and had good stability under the condition of dark storage at 4°C within a week or under the condition of dark storage at -20°C within one year. PRACTICAL APPLICATION: In this study, an efficient method for purification and refining of oleuropein by combinative technology off line of HSCCC-PHPLC based on dual wavelength was established. Oleuropein with the purity greater than 98.5% was macro-obtained via the technology. The highly purified oleuropein could be used to control the quality of olive products.

Lentiviral vector-mediated expression of C3 transferase attenuates retinal ischemia and reperfusion injury in rats.

AIMS: Our previous study showed that intravitreal delivery of self-complementary AAV2 (scAAV2)-mediated exoenzyme C3 transferase (C3) can attenuate retinal ischemia/reperfusion (I/R) injury. The current study investigated the neuroprotective effects of lentivirus (LV)-mediated C3 transgene expression on rat retinal I/R injury. MAIN METHODS: The LV encoding C3 and green fluorescent protein (GFP) together (LV-C3-GFP) or GFP only (LV-GFP) was intravitreally injected to SPRAGUE-DAWLEY rats. On day 5 post-intravitreal injection, eyes were evaluated by slit-lamp examination. The GFP expression on retina was confirmed by in vivo and ex vivo assessments. RhoA GTPase expression in retina was examined by western blot. Retinal I/R injury was generated by transiently increasing intraocular pressure (110 mmHg, 90 min). Eyes were then enucleated, and retinas processed for morphological analysis and TdT-dUTP terminal nick-end labeling (TUNEL) assay. KEY FINDINGS: No obvious inflammatory reactions or surgical complications were observed after intravitreal injection of LV vectors. There was a significant decrease of total RhoA GTPase level in the retina treated with LV-C3-GFP. Compared to the blank control group, LV-C3-GFP and LV-GFP did not affect the retinal thickness, cell density in ganglion cell layer (GCL), or numbers of apoptotic cells in retinal flat-mounts. In the LV-GFP-treated retinas, I/R decreased the retinal thickness and GCL cell density and increased apoptotic retinal cell numbers. LV-C3-GFP significantly protected against all these degenerative effects of I/R. SIGNIFICANCE: This study indicated that LV-mediated C3 transgene expression exhibits neuroprotective effects on the retinal I/R injury and holds potential as a novel neuroprotective approach targeting certain retinopathies.

Strategy for the separation of strongly polar antioxidant compounds from Lycium barbarum L. via high-speed counter-current chromatography.

In the separation of strongly polar antioxidant compounds from natural products using high-speed counter-current chromatography that is target-guided by 2,2-diphenyl-1-picrylhydrazyl high-performance liquid chromatography experimentation, low adsorption ability is encountered due to the strong polarity of the target compounds. In this study, a strategy of novel partition coefficient value calculation was proposed for overcoming this problem. The partition coefficient value was expressed as the ratio of the antioxidant activities of the upper phase and the lower phase. This strategy was used in high-speed counter-current chromatography with a hydrophilic organic/salt-containing aqueous two-phase system for bioassay-guided separation of strongly polar antioxidant compounds from Lycium barbarum L. The antioxidant activity was determined via the radical scavenging activity method using 2,2-diphenyl-1-picrylhydrazyl radicals. A hydrophilic organic/salt-containing aqueous two-phase system of 95% EtOH - sat. (NH4)2SO4 (1:1.8, v/v) was successfully used to separate Lycium barbarum L. extract. Five fractions were collected via high-speed counter-current chromatography separation. The antioxidant activity of the third fraction was the highest. Three compounds were separated via MCI gel column chromatography and Sephadex LH-20 column chromatography from the third fraction, and their antioxidant activities were determined. The antioxidant activities of the three compounds were higher than that of the third fraction. These results demonstrate that this strategy can be used to separate strongly polar antioxidant compounds from natural products.

Establishment and application of a method for screening the therapeutic drugs of ethanol-induced liver injury based on cellular metabonomics.

A drug-screening method to test the capacity of drugs to protect against ethanol-induced liver injury based on cellular metabonomics was established and applied in this study. It screens for the ability to protect against ethanol-induced liver injury by considering changes in the cellular metabolites of human normal liver L-02 cells subjected to ethanol treatment. This method considers cellular metabolites as the main analytical index, principal component analysis and orthogonal partial least squares discriminant analysis as the main multi- and megavariate data analysis methods, and vitamin C as the standard substance to determine the ability to protect against ethanol-induced liver injury. Ability to protect against ethanol-induced liver injury unit = [190 - 50× (14.318 - 10 × Y predictive value)1/2 ] × ability 1 µg/mL vitamin C. Olive leaf extract, Lycium barbarum L extract and fish roe peptide were screened using the established methods. Olive leaf OP phase had the strongest ability to protect against ethanol-induced liver injury, at 81.88. The value for L. barbarum L was 37.56. The fish roe peptide water phase was 63.07. All three have the ability to protect against ethanol-induced liver injury. The drug-screening method for ability to protect against ethanol-induced liver injury based on cell metabonomics is a fast, accurate and effective method for quantitative detection of ability to protect against ethanol-induced liver injury.

Chinese Eye Exercises and Myopia Development in School Age Children: A Nested Case-control Study.

Chinese eye exercises have been implemented in China as an intervention for controlling children's myopia for over 50 years. This nested case-control study investigated Chinese eye exercises and their association with myopia development in junior middle school children. Outcome measures were the onset and progression of myopia over a two-year period. Cases were defined as 1. Myopia onset (cycloplegic spherical equivalent ≤ -0.5 diopter in non-myopic children). 2. Myopia progression (myopia shift of ≥1.0 diopter in those who were myopic at baseline). Two independent investigators assessed the quality of Chinese eye exercises performance at the end of the follow-up period. Of 260 children at baseline (mean age was 12.7 ± 0.5 years), 201 were eligible for this study. There was no association between eye exercises and the risk of myopia-onset (OR = 0.73, 95%CI: 0.24-2.21), nor myopia progression (OR = 0.79, 95%CI: 0.41-1.53). The group who performed high quality exercises had a slightly lower myopia progression of 0.15 D than the children who did not perform the exercise over a period of 2 years. However, the limited sample size, low dosage and performance quality of Chinese eye exercises in children did not result in statistical significance and require further studies.

Long-term Efficacy of Trabeculectomy on Chinese Patients with Pigmentary Glaucoma: A Prospective Case Series Observational Study.

BACKGROUND: Though trabeculectomy is often performed on patients with medically refractive pigmentary glaucoma (PG), the clinical outcomes of surgical treatment on PG remain unknown. The aim of this study was to summarize the long-term efficacy and safety of trabeculectomy on PG. METHODS: This was a prospective case series observational study. Eighteen consecutive PG patients were followed up for 8 years after trabeculectomy from May 2006 to April 2007. Visual acuity (VA), best-corrected visual acuity (BCVA), slit lamp biomicroscopy, intraocular pressure (IOP) measurement, Humphrey visual field analysis (VFA), and stereoscopic funduscopy were performed on admission and every 6 months after the surgery. Postoperative IOP, VA, BCVA, VFA, adjunctive anti-glaucoma medication, treatment-related side-effects, changes in blebs, and main clinical findings in the anterior segment of PG were recorded and compared with the baseline. RESULTS: Eighteen PG eyes from 18 patients, with average preoperative IOP of 34.5 ± 4.7 mmHg (range: 21-47 mmHg, 1 mmHg=0.133 kPa) were enrolled in this study. All enrolled patients completed the follow-up visits and required examinations. Eight years after trabeculectomy, all surgical eyes (18/18) had satisfactory IOP control with an average of 13.7 ± 2.5 mmHg (range: 9-19 mmHg), which was significantly lower than baseline (P = 0.001). Majority (15/18) of the PG eyes had stable VA, BCVA, VFA, and optic disc cupping parameters. Functional blebs still existed in 12/18 of the PG eyes at the last follow-up visit. Unanimously, pigmentation in the anterior segment attenuated with time after surgical treatment. No severe side-effects were recorded in any of the surgical eyes. CONCLUSIONS: All surgical PG eyes in this study had satisfactory IOP control 8 years after the surgery with well-preserved visual function. The long-term efficacy and safety of trabeculectomy are promising in PG patients.

Efficacy of Chinese eye exercises on reducing accommodative lag in school-aged children: a randomized controlled trial.

PURPOSE: To evaluate the efficacy of Chinese eye exercises on reducing accommodative lag in children by a randomized, double-blinded controlled trial. METHODS: A total of 190 children aged 10 to 14 years with emmetropia to moderate myopia were included. They were randomly allocated to three groups: standard Chinese eye exercises group (trained for eye exercises by doctors of traditional Chinese medicine); sham point eye exercises group (instructed to massage on non-acupoints); and eyes closed group (asked to close their eyes without massage). Primary outcome was change in accommodative lag immediately after intervention. Secondary outcomes included changes in corrected near and distant visual acuity, and visual discomfort score. RESULTS: Children in the standard Chinese eye exercises group had significantly greater alleviation of accommodative lag (-0.10 D) than those in sham point eye exercises group (-0.03 D) and eyes closed group (0.07 D) (P = 0.04). The proportion of children with alleviation of accommodative lag was significantly higher in the standard Chinese eye exercises group (54.0%) than in the sham point eye exercises group (32.8%) and the eyes closed group (34.9%) (P = 0.03). No significant differences were found in secondary outcomes. CONCLUSION: Chinese eye exercises as performed daily in primary and middle schools in China have statistically but probably clinically insignificant effect in reducing accommodative lag of school-aged children in the short-term. Considering the higher amounts of near work load of Chinese children, the efficacy of eye exercises may be insufficient in preventing myopia progression in the long-term. TRIAL REGISTRATION: ClinicalTrials.gov NCT01756287.

[Clinical evaluation of a thermodynamic treatment system for meibomian gland dysfunction].

OBJECTIVE: To evaluate the effectiveness and safety of a single thermodynamic treatment system (LipiFlow) for meibomian gland dysfunction (MGD). METHODS: Retrospective series case study. Forty-eight subjects with meibomian gland dysfunction were analyzed before and after 12 minutes LipiFlow system treatment. All subjects were examined before, 4 weeks and 12 weeks after this treatment. Subjective symptoms, lipid layer thickness (LLT), expressible meibomian gland, tear break-up time, meibomian gland assessment and ocular surface staining were measured. Statistical analysis was performed using SPSS 11.5 software. Analysis included those independent sample two-tailed t-tests for comparison of the mean change from baseline to 4 weeks and baseline to 12 weeks after Lipiflow treatment. Statistically significant difference was based on α=0.05 (P<0.05). RESULTS: A total of 48 cases completed the 12-week follow-up. MGD patients with LipiFlow treatment had a significant reduction in the Ocular Surface Disease Index (OSDI) scores with 45.36±19.34 before treatment, 23.82±11.94 at 4 weeks (t=2.009, P=0.035) and 25.66±14.12 at 12 weeks (t=1.976 P=0.038). LipiFlow resulted in a higher number of expressible glands from 2.91±1.13 (baseline) to 6.27±2.37 (4 weeks, t=3.505, P<0.001) and 5.15±2.08 (12 weeks, t=2.004, P= 0.027) and change of secretion quality from 6.18±2.48 (baseline) to 13.55±3.46 (4 weeks,t=2.698,P=0.005) and 12.67±3.41 (12 weeks,t=2.403,P=0.009). In addition, a single thermodynamic treatment increase the LLT from (42.13±9.67)nm (baseline) to (59.02±16.39)nm (4 weeks, t=2.971, P=0.002) and (54.65±12.52)nm (12 weeks, t=2.021, P=0.021). The rate of partial blink was relieved from 0.37±0.30 (baseline) to 0.15±0.14 (4 weeks, t=3.428, P=0.035) and 0.12±0.13 (12 weeks, t=1.986, P=0.026). BUT was increased from (4.73± 2.34)s (baseline) to (9.32 ± 2.18)s (4 weeks, t=3.385, P<0.001) and (9.91 ± 3.01)s (12 weeks, t=3.253, P< 0.001). There were no unanticipated or serious device-related adverse events reported. Compared with baseline, there was no statistically significant difference in BCVA, corneal staining and intraocular pressure (P=0.141, 0.376, 0.421). CONCLUSION: The LipiFlow system was a safety and effectiveness treatment of MGD in the 12-week study.

Lycium barbarum polysaccharides promotes in vivo proliferation of adult rat retinal progenitor cells.

Lycium barbarum is a widely used Chinese herbal medicine prescription for protection of optic nerve. However, it remains unclear regarding the effects of Lycium barbarum polysaccharides, the main component of Lycium barbarum, on in vivo proliferation of adult ciliary body cells. In this study, adult rats were intragastrically administered low- and high-dose Lycium barbarum polysaccharides (1 and 10 mg/kg) for 35 days and those intragastrically administered phosphate buffered saline served as controls. The number of Ki-67-positive cells in rat ciliary body in the Lycium barbarum polysaccharides groups, in particular low-dose Lycium barbarum polysaccharides group, was significantly greater than that in the phosphate buffered saline group. Ki-67-positive rat ciliary body cells expressed nestin but they did not express glial fibrillary acidic protein. These findings suggest that Lycium barbarum polysaccharides can promote the proliferation of adult rat retinal progenitor cells and the proliferated cells present with neuronal phenotype.

Prevention of Selenite-Induced Cataratogenesis by Ginkgo biloba Extract (Egb761) in Wistar Rats.

OBJECTIVE: To investigate whether Ginkgo biloba extract retards selenite-induced cataractogenesis in Wistar rat pups. METHODS: On postpartum day eight, Group I rat pups (n = 12) received an intraperitoneal injection of physiological saline. Groups II and III rat pups (n = 12) received a subcutaneous injection of sodium selenite. Group III also received an intraperitoneal injection of G. biloba extract once daily on postpartum days 9-14. Both eyes of each pup were examined from postpartum day 16 up to day 30. After sacrifice, encapsulated pup lens were analyzed for mean activities of catalase, superoxide dismutase, glutathione peroxidase (GPx), glutathione S-transferase and glutathione reductase. In addition, the mean concentrations of reduced glutathione (GSH) and malondialdehyde were analyzed in samples of lens and serum. RESULTS: Dense lenticular opacification occurs 100% in Group II, but only minimal opacification occurs in three pups of Group III (25%), no opacification in 75% of Group III, none in Group I. Compared with Groups I and III, Group II rat showed lower lenticular antioxidant enzyme activity, lower level of GSH, and higher level of malondidehyde (mean ± standard deviation SD, p < 0.05 respectively). CONCLUSIONS: The treatment with G. biloba extract is effective against oxidative stress - a crucial factor of cataractogenesis in rat pups, possibly by preventing depletion of antioxidant enzymes and by inhibiting lipid peroxidation.

Effect of the regimen of Gaoshan Hongjingtian on the mechanism of poly (ADP-ribose) polymerase regulation of nuclear factor kappa B in the experimental diabetic retinopathy.

BACKGROUND: Poly(ADP-ribose) polymerase (PARP) plays an important role in the death of retinal capillary cells in diabetic retinopathy (DR) partly via its regulation of nuclear factor kappa B (NF-κB). The current study investigated the effect of the regimen of Gaoshan Hongjingtian (RG) on the mechanism of PARP regulation of NF-κB, and demonstrated the possible impact of the RG and Gaoshan Hongjingtian (Rhodiola sachalinensis, RS) on diabetic retinopathy. METHODS: Wistar rats were made diabetic by administering streptozotocin. They were then assigned to three groups at random. After 2 months, the three groups of these diabetic rats were treated with RS or RG, or untreated. Analyses of expression levels of PARP, NF-κB, and intercellular adhesion molecule-1 (ICAM-1) in the retinas of rats in different groups were performed by Western blotting and immunohistochemical assays, and mRNA levels of NF-κB and ICAM-1 were determined by real-time polymerase chain reaction (PCR). In addition, the basement membranes of capillaries in the rats' retinas were observed using electron microscopy, and diabetes-induced capillary degeneration (ghost pericytes and acellular capillaries) were quantitated. RESULTS: From the third month after the injection of streptozotocin, the diabetic rats were given daily RG, RS or tap water separately. The diabetic rats failed to gain weight compared with normal age-matched rats, whereas their glycated hemoglobin levels were significantly increased. After 5 months, the mRNA levels of NF-κB and ICAM-1 and the protein expression of PARP, NF-κB, and ICAM-1 were significantly increased in the retinas of diabetic rats in the untreated group compared with the nondiabetic controls. After 8 months, the number of degenerated retinal capillaries (ghost pericytes and acellular capillaries) was significantly increased in the diabetic rats in the untreated group compared with normal age-matched rats. RG and RS inhibited diabetes-induced over-expression of PARP, NF-κB, and ICAM-1 in the retinas of diabetic rats at the end of 5-month diabetic duration. Treatment using RG and RS significantly inhibited increases in the number of acellular capillaries and pericyte ghosts and suppressed the basement membrane thickening in the retinas of rats with diabetes for 8 months compared with the control diabetic rats. CONCLUSIONS: These results indicate that PARP plays an important role in the pathogenesis of diabetic retinopathy. RS and RG may have acted on the mechanism of PARP regulation of NF-κB, which suppressed the expression of NF-κB and ICAM-1, and led to the inhibition of retinal capillary degeneration.