Combination of Lactobacillus fermentum NS9 and aronia anthocyanidin extract alleviates sodium iodate-induced retina degeneration.

It is important to explore the effective approaches to prevent dry age-related macular degeneration (AMD). In this study, significantly decreased full-field electroretinograms wave amplitudes and disordered retina structures were detected in rat retinas of sodium iodate induced dry AMD model. Six a- and b-wave amplitudes and the antioxidant activities were significantly increased, and the outer nuclear layer thickness was significantly improved in the rat retinas treated with the combination of Lactobacillus fermentum NS9 (LF) and aronia anthocyanidin extract (AAE) compared with the model. The effects were much better than the treatment with AAE alone. The proteomics analysis showed the expressions of α-, ß- and γ-crystallins were increased by 3-8 folds in AAE treated alone and by 6-11 folds in AAE + LF treatment compared with the model, which was further confirmed by immuno-blotting analysis. Analysis of gut microbial composition indicated that higher abundance of the genus Parasutterella and species P. excrementihominis was found in the AAE + LF treatment compared with the other groups. The results indicated that the combined treatment of AAE + LF is a potential way to prevent the retina degeneration which is significantly better than the AAE treated alone.

Physicochemical and nutritional properties of yogurt emulsion with lycopene during chilled storage.

Lycopene is a highly potent antioxidant that is prevalent among dietary carotenoids. However, its use in food formulations is restricted due to its poor water-solubility and proneness to oxidation. The aim of this research was to encapsulate lycopene in yogurt using emulsion technology for improving its stability during processing and storage, in order to diversify a widely consumed food product and enhance its nutritional value. Confocal laser microscopy data showed that the incorporation of oil droplets with emulsification did not have a negative effect on the formation and microstructure of yogurt. Syneresis of lycopene-fortified yogurt samples was approximately twice as high compared with plain yogurt at day 7; the ability to retain water was significantly improved with storage time for all emulsified samples. Additionally, storage reduced the Turbiscan Stability Indices (TSI) for all yogurt samples, which suggests that physical stability improved at 4 °C. Emulsification resulted in increased oxidation levels due to increased oil content. This effect was ameliorated by lycopene encapsulation, which effectively protected corn oil from oxidation and prevented degradation. This study indicates that emulsification is a promising method for lycopene encapsulation and can be used for developing yogurt with desirable nutritional properties.

Isolation and identification of antioxidative peptides from crocodile meat hydrolysates using silica gel chromatography.

Crocodiles are cultured in large numbers in Asia and other places in order to protect wild resources and meet the needs of human life. In this study, crocodile (Crocodylus siamensis) meat proteins were extracted and hydrolyzed into peptides, their antioxidant peptides were isolated and purified by silica gel chromatography and identified by LC/MS. Crocodile meat proteins were optimally extracted with water and hydrolyzed by papain based on the degree of hydrolysis and antioxidant activity. The hydrolysates were fractionated by ultrafiltration into 3 kDa, 3-30 kDa, and ≥ 30 kDa fractions. The 3 kDa fraction showed most antioxidant activity of the hydrolysates. Its active peptides were separated by silica gel column chromatography and purified by silica gel TLC, based on TLC bio-autographic assays of the activity. Four highly active peptides were identified by LC/MS as SSLTIQFVEGQFVDSYDPTIENTFTK, VPPHIY, VAPEEHPVLLTEAPLNPK, and RNGLPGPIGPAG. The identified peptides were synthesized and showed 50% free radical scavenging activities at 1.0 mg/mL, equal or higher to ascorbic acid at 0.5 mg/mL, in both DPPH and ABTS assays. The results indicated that the 3 kDa hydrolyzed peptides of crocodile meat had high antioxidant activity and the active peptides can be effectively separated and purified by silica gel column chromatography and TLC.

Simultaneous extraction and separation of oil, proteins, and glucosinolates from Moringa oleifera seeds.

Moringa oleifera is a worldwide cultivated edible and medicinal plant. Its seeds are rich in oil, proteins, and glucosinolates. A practical method was developed to simultaneously extract and separate the three groups of substances from M. oleifera seeds. Smashed seed material was loaded into columns with petroleum ether: ethanol 8:2 (PE-ethanol) and eluted sequentially with 4.8-fold PE-ethanol to extract oil, and 10.8-fold water to extract proteins and glucosinolates. More than 95% of oil, proteins, and glucosinolates were extracted. The extracts were separated automatically into ether (oil) phase and ethanol aqueous phase. The latter was further separated into proteins and glucosinolates by 70% ethanol precipitation. The main glucosinolate was identified by LC-MS as GLC (4-α-rhamnopyranosyloxy-benzyl glucosinolate). After purification, 22.3 g refined oil, 33.0 g proteins, and 5.5 g purified GLC from 100 g M. oleifera seeds were obtained. This study provides a simple and high-efficient method to utilize M. oleifera seeds.

Preparation of peroxidase and phenolics using discarded sweet potato old stems.

Sweet potato (Ipomoea batatas L.) is the sixth most important food crop in the world. The industry discarded huge amount of sweet potato stems, rich of peroxidases and phenolics. A simple procedure was developed to make peroxidases and phenolics from sweet potato old stems. Dried stem powder was loaded into columns with water and eluted sequentially with water and 50% ethanol. Peroxidases (91%) were extracted in 5.5-fold water extracts and 87% phenolics were extracted in 4.4-fold ethanol extracts. Purified peroxidases powder was yielded at 3.1 g (8.6 unit/mg) per kilogram stems by PEG6000/Na2SO4 aqueous two-phase purification from the water extracts (93.2% recovery), followed by ethanol precipitation and vacuum freeze-drying. The purified peroxidase had high activity in transforming tea catechins into theaflavins. Phenolics powder containing 43% phenolics and 27% flavonoids was yielded at 76.9 g per kilogram stems after vacuum-concentrating the ethanol extracts. This method can make valuable functional products using the sweet potato waste.

Simplified recovery of enzymes and nutrients in sweet potato wastewater and preparing health black tea and theaflavins with scrap tea.

The industry discards generous organic wastewater in sweet potato starch factory and scrap tea in tea production. A simplified procedure to recover all biochemicals from the wastewater of sweet potato starch factory and use them to make health black tea and theaflavins from scrap green tea was developed. The sweet potato wastewater was sequentially treated by isoelectric precipitation, ultrafiltration and nanofiltration to recover polyphenol oxidase (PPO), ß-amylase, and small molecular fractions, respectively. The PPO fraction can effectively transform green tea extracts into black tea with high content of theaflavins through the optimized fed-batch feeding fermentation. The PPO transformed black tea with sporamins can be used to make health black tea, or make theaflavins by fractionation with ethyl acetate. This work provides a resource- and environment-friendly approach for economically utilizing the sweet potato wastewater and the scrap tea, and making biochemical, nutrient and health products.

Extraction and preparation of high-aroma and low-caffeine instant green teas by the novel column chromatographic extraction method with gradient elution.

The lack of aroma and natural taste is a critical problem in production and consumption of instant green teas. A method to prepare instant green teas high in-natural-aroma and low-caffeine by the novel column chromatographic extraction with gradient elution is reported. This method simultaneously extracted aroma (or volatile) and non-aroma compounds from green tea. Green tea was loaded into columns with 2.0-fold of petroleum ether (PE): ethanol (8:2). After standing for 3 h until the aroma compounds dissolved, the column was sequentially eluted with 3.0-fold 40% ethanol and 3.5-fold water. The eluant was collected together and automatically separated into PE and ethanol aqueous phases. The aroma extracts was obtained by vacuum-evaporation of PE phase at 45 °C. The ethanol aqueous phase was vacuum-concentrated to aqueous and partially or fully decaffeinated with 4% or 9% charcoal at 70 °C. A regular instant green tea with epigallocatechin-3-gallate: caffeine of 3.5:1 and a low-caffeine instant green tea (less than 1% caffeine) with excellent aroma and taste were prepared, by combining the aroma and non-aroma extracts at a 1:10 ratio. This work provides a practical approach to solve the low-aroma and low-taste problems in the production of high quality instant green teas.

Comprehensive utilization of activated sludge for the preparation of hydrolytic enzymes, polyhydroxyalkanoates, and water-retaining organic fertilizer.

The urban wastewater treatment industry produces a large amount of excess activated sludge which is mainly composed of microbial biomass and costly to be disposed. In this research, a comprehensive utilization of activated sludge was developed by sequentially extracting hydrolytic enzymes and polyhydroxyalkanoates (PHAs), and the residue was used to prepare water-retaining organic fertilizer. The sludge was extracted with fourfold H2O-containing 1% Triton X-100 with the yield of 66.7% protease activity. The enzyme solution was precipitated in 80% acetone and vacuum dried at 40°C at the dried enzyme yield of 2.4 g/kg wet sludge. The enzyme product contains collagenase, lipase, amylase, and cellulase activities, which are good compound enzymes to feed. The PHAs were extracted with 30% sodium hypoclorite:chloroform (1:3). The PHA solution was decolored and dried, and pure white PHAs were obtained at the yield of 70.1 g/kg wet sludge. The residue was used to prepare water-retaining organic fertilizer at the optimal condition. The fertilizer absorbs 131.3-fold distilled water and had good performance in water retention and can effectively slow down the loss of soil moisture when added into soil. This work provides a simple and practical approach for comprehensive utilizing activated sludge with significant economic benefits.

Expression of nattokinase in Escherichia coli and renaturation of its inclusion body.

Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8IU/mg and 79.3IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution.

Segmental ureteroileal conduit resection for the treatment of distal upper urinary tract recurrence of bladder cancer following cystectomy.

Segmental ureterectomy is less invasive than radical nephroureterectomy and results in nephron preservation and satisfactory tumor control. This study was to determine the feasibility of segmental ureteroileal conduit resection (SUICR) for patients with distal upper urinary tract recurrence of bladder cancer following radical cystectomy. Four patients with high-grade distal upper urinary tract recurrence underwent SUICR 15-108 months after radical cystectomy. The surgical technique details of SUICR, operative results, and follow-up outcomes are reported. The median operation time was 280 min, and estimated blood loss was less than 100 mL. One patient suffered from ileus 5 days after surgery and was managed conservatively. Histopathologic evaluation showed high-grade stages pTa-pT1 diseases for these patients, and ureteral margins were all negative. No patient suffered from tumor recurrence, with a median follow-up of 39 months. SUICR preserved the ipsilateral renal unit and conformed to oncological principles during surgery. The oncological outcome was satisfactory for these properly selected patients. This technique provides a valid alternative to nephroureterectomy for patients with imperative indications and high-grade upper urinary tract recurrence of bladder cancer following radical cystectomy.

Simultaneous column chromatographic extraction and purification of abscisic acid in peanut plants for direct HPLC analysis.

Abscisic acid (ABA), a universal signaling molecule, plays important roles in regulating plant growth, development and stress responses. The low contents and complex components in plants make it difficult to be accurately analyzed. A novel one-step sample preparation method for ABA in plants was developed. Fresh peanut (Arachis hypogaea) plant materials were fixed by oven-drying, microwave drying, boiling or Carnoy's fixative, and loaded onto a mini-preparing column. After washed the impurities, ABA was eluted with a small amount of solvent. ABA in plant materials was completely extracted and purified in 2mL solution and directly analyzed by HPLC, with a 99.3% recovery rate. Multiple samples can be simultaneously prepared. Analyses using this method indicated that the endogenous ABA in oven-dried peanut leaves increased 20.2-fold from 1.01 to 20.37µgg(-1) dry weight within 12h and then decreased in 30% polyethylene glycol 6000 treated plants, and increased 3.34-fold from 0.85 to 2.84µgg(-1) dry weight in 5 days and then decreased in soil drought treated plants. The method combined the column chromatographic extraction and solid-phase separation technologies in one step and can completely extracted plant endogenous ABA in a purified and highly concentrated form for direct HPLC analysis.

Screening, separating, and completely recovering polyphenol oxidases and other biochemicals from sweet potato wastewater in starch production.

Polyphenol oxidase (PPO) has multiple functions, and the lack of commercially available enzyme sources limits its widespread application in various industries. An accurate PPO assay was developed by HPLC determination of the substrate oxidation. Resources screening indicated that sweet potato (Ipomoea batatas L.) wastewater in starch production has high PPO activity. A procedure was developed for separately recovering PPO, ß-amylase, sporamins, and small molecular nutrients (SMNs) from sweet potato wastewater. The wastewater was adjusted to pH 3.5 to precipitate PPO, and then adjusted to 50 % acetone to precipitate ß-amylase and further to 80 % acetone to precipitate sporamins. The SMNs were obtained after acetone recovery. Purified powders of 4.3 × 10(5) units of PPO, 4.0 × 10(6) units of ß-amylase, 8.70 g sporamins, and 20.2 g SMNs were obtained from the wastewater of 1 kg sweet potato. More than 50 million tons of sweet potato is used for starch production annually around the world. Through this simple procedure, huge amount of biochemical resources can be recovered from the wastewater, which greatly increases the economic value of the crop and saves the environment.

Analysis of trace metals and perfluorinated compounds in 43 representative tea products from South China.

UNLABELLED: Six trace metals (Cd, Pb, Cr, Cu, Zn, and Mn) and 2 perfluorinated compounds (PFCs), perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), were analyzed in 43 representative tea products (including 18 green, 12 Oolong, and 13 black teas) from 7 main tea production provinces in China, using the atomic absorption spectrophotometer for trace metals analysis and HPLC-MS/MS for PFOS and PFOA analysis. The average contents of the 3 essential metals Mn, Cu, and Zn ions in the tea samples were 629.74, 17.75, and 37.38 mg/kg, whereas 3 toxic metals Cd, Cr, and Pb were 0.65, 1.02, and 1.92 mg/kg, respectively. The contents of heavy metals in the 3 types of tea were in the order of black tea > Oolong tea > green tea. Both PFOS and PFOA contents were low and PFOA content was higher than PFOS in the tea samples. The highest concentration of PFOA was 0.25 ng/g dry weight found in a Hunan green tea. The Principal component analysis was performed with the trace metals and PFCs to analyze the relationships of these indices. The results showed that black teas had higher trace metals and PFCs than green and Oolong teas, and the teas from Hunan and Zhejiang provinces had higher Pb and Cr than others. PRACTICAL APPLICATION: This paper reports trace metals, and perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in wide range of tea products produced in the south China area. This paper also warns the low PFOS and PFOA pollution in tea.

Modulation effects of cordycepin on the skeletal muscle contraction of toad gastrocnemius muscle.

Isolated toad gastrocnemius muscle is a typical skeletal muscle tissue that is frequently used to study the motor system because it is an important component of the motor system. This study investigates the effects of cordycepin on the skeletal muscle contractile function of isolated toad gastrocnemius muscles by electrical field stimulation. Results showed that cordycepin (20 mg/l to 100 mg/l) significantly decreased the contractile responses in a concentration-dependent manner. Cordycepin (50 mg/l) also produced a rightward shift of the contractile amplitude-stimulation intensity relationship, as indicated by the increases in the threshold stimulation intensity and the saturation stimulation intensity. However, the most notable result was that the maximum amplitude of the muscle contractile force was significantly increased under cordycepin application (122±3.4% of control). This result suggests that the skeletal muscle contractile function and muscle physical fitness to the external stimulation were improved by the decreased response sensitivity in the presence of cordycepin. Moreover, cordycepin also prevented the repetitive stimulation-induced decrease in muscle contractile force and increased the recovery amplitude and recovery ratio of muscle contraction. However, these anti-fatigue effects of cordycepin on muscle contraction during long-lasting muscle activity were absent in Ca2+-free medium or in the presence of all Ca2+ channels blocker (0.4 mM CdCl2). These results suggest that cordycepin can positively affect muscle performance and provide ergogenic and prophylactic benefits in decreasing skeletal muscle fatigue. The mechanisms involving excitation-coupled Ca2+ influxes are strongly recommended.

One-step column chromatographic extraction with gradient elution followed by automatic separation of volatiles, flavonoids and polysaccharides from Citrus grandis.

Citrus grandis Tomentosa is widely used in traditional Chinese medicine and health foods. Its functional components include volatiles, flavonoids and polysaccharides which cannot be effectively extracted through traditional methods. A column chromatographic extraction with gradient elution was developed for one-step extraction of all bioactive substances from C. grandis. Dried material was loaded into a column with petroleum ether: ethanol (8:2, PE) and sequentially eluted with 2-fold PE, 3-fold ethanol: water (6:4) and 8-fold water. The elutes was separated into an ether fraction containing volatiles and an ethanol-water fraction containing flavonoids and polysaccharides. The later was separated into flavonoids and polysaccharides by 80% ethanol precipitation of polysaccharides. Through this procedure, volatiles, flavonoids and polysaccharides in C. grandis were simultaneously extracted at 98% extraction rates and simply separated at higher than 95% recovery rates. The method provides a simple and high-efficient extraction and separation of wide range bioactive substances.

Effects of cordycepin on Y-maze learning task in mice.

Cordycepin (3'-deoxyadenosine) is the major bioactive component of Cordyceps militaris that has been widely used in oriental countries as a Traditional Chinese Medicine and healthy food for preventing early aging, improving physical performance and increasing lifespan. Cordyceps militaris extracts other than cordycepin have been reported to improve cognitive function. Although cordycepin is one of the most utilized Cordyceps militaris components, it remains unknown whether cordycepin could improve learning and memory. Here we investigated effects of cordycepin on learning and memory in healthy and ischemic mice using Y-maze test. We found that oral cordycepin administration at dose of 10 mg/kg significantly improved Y-maze learning performance both in healthy and ischemic mice. However, cordycepin at dose of 5 mg/kg enhanced Y-maze learning only in ischemic mice but not healthy mice. In this study, simultaneously, we found that orally administrated cordycepin significantly decreased the neuronal loss induced by ischemia in hippocampal CA1 and CA3 regions. Collectively, our results can provide valuable evidence that cordycepin may act as a nootropic product or potential clinical application in improving cognitive function of patients with ischemic stroke in the future.

New and highly efficient column chromatographic extraction and simple purification of camptothecin from Camptotheca acuminata and Nothapodytes pittosporoides.

INTRODUCTION: Camptothecin, a widely used natural anti-cancer drug, is difficult to extract and purify effectively from plants. OBJECTIVE: To develop new and highly efficient extraction and purification methods for analysis and production of camptothecin from leaves and fruits of Camptotheca acuminata and Nothapodytes pittosporoides roots. METHODS: Dried materials were loaded in empty columns with fivefold 60% ethanol for leaves or 70% ethanol for fruits of C. acumnata, and sixfold 70% ethanol for N. pittosporoides roots. The columns were eluted with the same solvents at room temperature. Eluent was collected as extraction solution. Extraction solution from leaves and fruits of C. acuminata was vacuum-evaporated to remove ethanol, precipitated at pH 8.0 to remove alkaline insolubles and fractionated with chloroform at pH 3.0, which yields a crude product with 70% purity. Extraction solution from N. pittosporoides roots was concentrated to 1/10 volume and precipitated at pH 3.0, which yields a crude product with 60% purity. All crude products were purified by crystallisation. All steps were monitored by HPLC. RESULTS: Camptothecin was extracted from the three plant materials at a 98% rate with 15- or 18-fold solvent for content analysis, or at a 97% rate with five- or sixfold solvent for production. All crude products were purified to 98%. The overall recovery rates of camptothecin from plant materials to purified products reached 92% or higher. CONCLUSION: The new procedures are simple and highly efficient, and have multiple advantages for quantitative analysis and large production of camptothecin from plants.

Cordycepin suppresses excitatory synaptic transmission in rat hippocampal slices via a presynaptic mechanism.

AIMS: Cordycepin plays an important role in modulating the function of central nervous system (CNS). However, the modulating mechanism is poorly understood. Excitatory synaptic transmission, the essential process in brain physiology and pathology, is critical in the signal integration activities of the CNS. To further understand the effects of cordycepin on CNS, we investigated the effects of cordycepin on excitatory synaptic transmission in the CA1 region of rat hippocampal slices. METHODS: The effects of cordycepin on excitatory synaptic transmission were investigated by using in vitro field potential electrophysiology and whole-cell patch clamp techniques. RESULTS: Cordycepin significantly decreased the amplitudes of field excitatory postsynaptic potentials (fEPSPs) elicited in the CA1 by stimulation of the Schaffer-commissural fibers. And the reduction in fEPSPs amplitude was associated with an increase in the paired-pulse facilitation. Cordycepin also suppressed α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor-mediated responses but did not directly affect AMPA receptors and NMDA receptors. Furthermore, quantal analysis revealed that cordycepin decreased the frequency but not amplitude of miniature spontaneous excitatory postsynaptic currents. CONCLUSIONS: These results demonstrate that cordycepin suppresses excitatory synaptic transmission by decreasing the excitatory neurotransmitter release presynaptically, which provides an evidence for the novel potential mechanism of cordycepin in modulating the function of CNS.

Cloning a peanut resveratrol synthase gene and its expression in purple sweet potato.

A resveratrol synthase gene was cloned from the peanut plant (Arachis hypogaea) by RT-PCR and was transformed into purple sweet potato (Ipomoea batatas) by Agrobacterium-mediated transformation. Stem sections were infected with bacterial solution of OD(600) = 0.4 for 20 min and then cocultured for 2 days. Infected explants were cultured on MS media containing 50 mg/l kanamycin, 0.02 mg/l NAA and 1 mg/l 6-BA for bud induction or containing 75 mg/l kanamycin, 1.0 mg/l NAA and 0.1 mg/l 6-BA for root formation. The bud and root induction rates were 37.5 and 25.0%, respectively. 105 regenerated plants were obtained, with 11 positive plants by PCR and Southern blotting analyses. A high level of resveratrol glucoside (340 µg/g dry weight), but no resveratrol, was detected in the transformed plants by HPLC. This study also provides a stable genetic transformation and plant regeneration method for metabolic modification of purple sweet potato.

Anatomic study of popliteus complex of the knee in a Chinese population.

This study explored the anatomic relationship of the popliteus complex (PC) and collected anatomic data of PC in a Chinese population. The anatomic study was performed using 81 formalin-fixed knees. The femoral attachment of the popliteus tendon could be classified into three types with respect to femoral attachment of the lateral collateral ligament. The popliteofibular ligament presented as ligament (87.7%) or fascia (12.3%), originating from the musculotendinous junction of the popliteus muscle or just proximal to it. Given the great variability of the posterolateral structures, reconstructive surgeons should design a method based on individual anatomic features, rather than a fixed reconstructive surgery.