Hydrolysed collagen from Lates calcarifer skin: its acute toxicity and impact on cell proliferation and collagen production of fibroblasts

Acute toxicity in Wistar rat and the impact of hydrolysed collagen (HC) from seabass skin on in vitro cell proliferation and collagen production were studied using L929 fibroblasts. HC was rich in glycine (326 residue/1000 residue) and imino acids (196 residue/1000 residue). MALDI mass spectrum of HC showed several low MW peptides with MW range of 1050–1330 Da as the major components. Based on acute oral cytotoxicity test in Wistar rat, HC was considered as safe with LD₅₀ value higher than 5000 mg kg^?1 body. HC could promote L929 cell growth, especially when used in combination with vitamin C (VitC) at a ratio of 2:1. HC/VitC (2:1) mixture also exhibited the higher enhancement effect on collagen production of L929 cells, compared with HC or VitC alone. Thus, HC could be a promising candidate for biological applications, especially in combination with VitC, as nutraceuticals for skin care.     

INVESTIGATION OF THE MECHANICS OF SINGLE TOMATO FRUIT CELLS

The mechanical behavior of single tomato fruit cells has been characterized using high strain‐rate microcompression testing. Single cells isolated by gentle washing from inner pericarp tissue were compressed to a wide range of deformations at a speed of 1500 µm/s, and then released. The cells were larger than any tested previously by microcompression, and had very low initial turgor. Force‐deformation data were modeled to find cell wall material properties, assuming water loss during compression could be neglected because of fast compression. Repeat compression‐release experiments were conducted to discover when cell deformation was no longer recoverable upon release. Cells from three commercially grown tomatoes were elastic to deformations of just over 11%. The elastic moduli of the cell walls were found by modeling to be 30 to 80 MPa, significantly lower than suspension‐cultured cell walls. The cell walls yielded at about 2% wall strain. High‐speed compression testing is a powerful tool for studying low turgor cells, such as those found during ripening.     

In vitro cultured human skin cells as alternatives to animals for skin irritancy screening

In the last few years a lot of attention has been paid to the development of the in vitro models which would substitute for animals in cutaneous irritancy studies. These models explore either organ or explant cultures using freshly excised skin or serial cultures of isolated skin cells (epidermal keratinocytes or dermal fibroblasts). The organ or explant models are suitable only for short exposures of skin samples to the compounds tested and the use of it will always be restricted by the limited availability of fresh human skin. The model that uses submerged cultures of keratinocytes or fibroblasts permits the production of a large number of cells, and permits large scale toxicity screening tests with many substances, that can be applied in a broad concentration range. Since the stratum corneum is absent in conventional (submerged) keratinocyte culture systems, this model is mainly suited for testing of water soluble compounds and it is less suitable for poorly soluble compounds and for topical products consisting of complex formulations which are made of active ingredients and their vehicles. This shortcoming can be overcome by using ‘organotypic cultures’in which keratinocytes are grown at the air-liquid interface on a suitable dermal substrate. Under these conditions, the culture forms a multilayered epidermis showing an overall structure which resembles that of a native epidermis. The presence of a coherent stratum corneum layer in these cultures permits the application of potential irritants at concentrations and in formulations as applied in vivo. For the evaluation of toxicity a number of tests have already been developed: assessment of cell viability, changes in cell morphology, modulation of cell proliferation and differentiation, monitoring of membrane damage, the measurements of the uptake or incorporation of radioactive precursors, establishment of the modulation of cell metabolism, determination of the release of inflammatory mediators, etc. All these in vitro techniques are still in a state of validation as far as their predictive value for in vivo skin irritancy is concerned.     

Antioxidative and Anti-Inflammatory Neuroprotective Effects of Astaxanthin and Canthaxanthin in Nerve Growth Factor Differentiated PC12 Cells

ABSTRACT: Nerve growth factor differentiated PC12 cells were used to examine the antioxidative and anti‐inflammatory effects of astaxanthin (AX) and canthaxanthin (CX). PC12 cells were pretreated with AX or CX at 10 or 20 μM, and followed by exposure of hydrogen peroxide (H₂O₂) or 1‐methyl‐4‐phenylpyridinium ion (MPP⁺) to induce cell injury. H₂O₂ or MPP⁺ treatment significantly decreased cell viability, increased lactate dehydrogenase (LDH) release, enhanced DNA fragmentation, and lowered mitochondrial membrane potential (MMP) (P < 0.05). The pretreatments from AX or CX concentration‐dependently alleviated H₂O₂ or MPP⁺‐induced cell death, LDH release, DNA fragmentation, and MMP reduction (P < 0.05). Either H₂O₂ or MPP⁺ treatment significantly increased malonyldialdehyde (MDA) and reactive oxygen species (ROS) formations, decreased glutathione content, and lowered glutathione peroxidase (GPX) and catalase activities (P < 0.05). The pretreatments from AX or CX significantly retained GPX and catalase activities, and decreased MDA and ROS formations (P < 0.05). H₂O₂ or MPP⁺ treatment significantly decreased Na⁺‐K⁺‐ATPase activity, elevated caspase‐3 activity and levels of interleukin (IL)‐1, IL‐6, and tumor necrosis factor (TNF)‐α (P < 0.05); and the pretreatments from these agents significantly restored Na⁺‐K⁺‐ATPase activity, suppressed caspase‐3 activity and release of IL‐1, IL‐6, and TNF‐α (P < 0.05). Based on the observed antioxidative and anti‐inflammatory protection from AX and CX, these 2 compounds were potent agents against neurodegenerative disorder.     

Electrospinning of single and multilayered scaffolds for tissue engineering applications

Optimized electrospinning conditions were applied to produce single and multilayered (ML) scaffolds composed of polycaprolactone, collagen and elastin. The ML scaffold was cross-linked with glutaraldehyde to increase the stability. Morphological and structural characteristics of the scaffolds were measured by SEM and FTIR analyses. Results revealed that polymers combined to each other well and uniform fibers were obtained with the diameters ranging from 156 ± 53 to 1536 ± 293 nm. Contact angle measurements were performed to investigate the hydrophilic character of each structure. It was observed that incorporation of the natural polymers into the blends increased the hydrophilicity. Mechanical tests proved that collagen contributed to fabricate stiffer structures while elastin provided more elasticity. Biocompatibility of the scaffolds was examined by SEM analysis and WST-1 test with mouse fibroblast cells (L929) in vitro. Results exhibited that the addition of natural polymers increased the cell growth, and none of the single and ML scaffolds presented cytotoxic effect.     

Comparison of Axial and Radial Compression Tests for Determining Elasticity Modulus of Potatoes

The effect of sample size, loading rate, and compression level on the determination of tangent modulus of elasticity of potatoes in axial (E_a) and radial (E_r) compression tests was studied. Cylindrical potato samples of 10, 15, and 20 mm diameter with length-to-diameter (L/D) ratio 1, 1.5, and 2 and loading rates of 50, 100, 200, 300, and 400 mm/min were used. A third-degree polynomial best described the force-deformation (F-D) plot of the test specimens (R²?=?0.98 to 0.99) in both tests. In view of the non-linearity of the F-D plot, E_a and E_r were calculated corresponding to 10, 20, and 30% compression. The values of E_a and E_r obtained for various combinations of sample size and compression level had coefficient of variation (CV) of about 24 and 8%, respectively, indicating relatively greater influence of experimental conditions in axial than radial compression testing. Apparently, E_a increased with an increase in compression level and loading rate whereas E_r exhibited inconsistent trend. The combined effects of sample size and compression level accounted for about 93 and 20% of the changes in E_a and E_r, respectively signifying the minimal effects of these parameters in radial compression testing. Similarly, the loading rate and compression level accounted for about 96 and 28% of the changes in E_a and E_r, respectively. The results of this article revealed that determination of modulus of elasticity of potatoes in axial compression testing was significantly influenced by testing conditions and sample size, whereas radial compression testing appeared to be independent of testing conditions and sample size.     

Anti-obesity effect of a standardised ethanol extract from Curcuma longa L. fermented with Aspergillus oryzae in ob/ob mice and primary mouse adipocytes

BACKGROUND: We examined the anti‐obesity effect of fermented Curcuma longa L. (turmeric) standardised ethanol extract (FTE) in the C57BL/6J ob/ob mouse model. Mice were fed a chow diet containing FTE (0, 200, or 500 mg kg^?1 body weight) for 9 weeks. RESULTS: Supplementation with FTE significantly reduced body weight gain and retroperitoneal and epididymal adipose tissue weights compared to the ob/ob control group. Additionally, total cholesterol and triglyceride levels in serum and liver were significantly decreased in FTE‐200 and FTE‐500 groups when compared to those of the ob/ob control group, whereas the high‐density lipoprotein‐cholesterol level was significantly increased. The levels of serum adiponectin as well as mRNA expression of lipases, such as hormone sensitive lipase and adipose triglyceride lipase, were clearly increased. In primary adipocytes of C57BL/6J mice, FTE treatment caused a significant increase glycerol release and hormone sensitive lipase levels and decreased perilipin A levels. CONCLUSION: These results suggest that supplementation of FTE has potent anti‐obesity effects by controlling body weight, fat mass, serum lipids, and hepatic lipids. Moreover, FTE could be considered a potential resource for the treatment of obesity through its promotion of lipolysis via the protein kinase A pathway. Copyright © 2012 Society of Chemical Industry     

Dietary exposure of Hong Kong adults to fatty acid esters of 3-monochloropropane-1,2-diol

A total of 290 individual food samples were collected in Hong Kong, China, for 3-monochloropropane-1,2-diol (3-MCPD) fatty acid esters analysis. Most samples were processed food and in ready-to-eat form. The results show that the levels of 3-MCPD fatty acid esters were high in biscuits, fats and oils, snacks and Chinese pastry with mean bound 3-MCPD levels of 440, 390, 270 and 270 μg kg^?1, respectively. The dietary exposures to bound 3-MCPD of average and high adult consumers were estimated to be 0.20 and 0.53 μg kg bw^?1 day^?1, respectively. The primary toxicological concern of 3-MCPD fatty acid esters is its potential to release 3-MCPD in vivo during digestion in the gastrointestinal tract. 3-MCPD would affect the kidney, the central nervous system and the male reproductive system of rats. Assuming that 100% of the 3-MCPD was released from 3-MCPD fatty acid esters by hydrolysis in the digestive system, the dietary exposures to 3-MCPD for average and high adult consumers were only 10% and 26% of the provisional maximum tolerable daily intake (PMTDI) of 3-MCPD established by the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives (JECFA) (2 μg kg bw^?1 day^?1), respectively. The results suggest that both average and high adult consumers are unlikely to experience major toxicological effects of 3-MCPD.     

Pulsed electric fields- and ultrasound-assisted green extraction of valuable compounds from Origanum vulgare L. and Thymus serpyllum L.

This work compared the effects of pulsed electric fields (PEF) and ultrasound (US) technologies on the extent of cell disintegration of two Mediterranean herb tissues (Origanum vulgare L., Thymus serpyllum L.), as well as on the extractability of phenolic compounds during the subsequent hydroalcoholic extraction (0%–50% ethanol in water, v/v) for up to 4 h. The rate of phenolic compounds extraction decreased with time and was predicted rather satisfactorily (R² = 0.898–0.989) by the Peleg’s model. The application of either PEF or US treatment prior to solid–liquid extraction (SLE) has the potential to reduce duration and concentration of ethanol to achieve the same recovery yield of phenolic compounds. Under optimised PEF (3 kV cm⁻¹, 10 kJ kg⁻¹) and US (400 W, 20 min) treatment conditions, the extracts obtained from either PEF or US pretreated herb samples showed higher total phenolic yield (36% on average) and antioxidant activity (FRAP) (36% on average) as compared to the control extraction, especially when 25% ethanol was used as a solvent. GC/MS analyses revealed no evidence of degradation of individual phenolics due to either PEF or US application.     

Labisia pumila extract protects skin cells from photoaging caused by UVB irradiation

Labisia pumila (Myrsinaceae), known as “Kacip Fatimah,” has been used by many generations of Malay women to induce and facilitate child birth as well as a post partum medicine. However, its topical application on skin has not been reported yet. In this study, we have focused on the anti-photoaging effects of L. pumila. Extract of L. pumila was first analyzed for their antioxidant activities using DPPH (2,2-diphenyl-1-picrylhydrazyl) since UV irradiation is a primary cause of reactive oxygen species (ROS) generation in the skin. The 50% free radical scavenging activity (FSC₅₀) of L. pumila extract was determined to be 0.006%, which was equal to that produced by 156 μM ascorbic acid. TNF-α and cyclooxygenase (COX-2) play a primary role in the inflammation process upon UV irradiation and are known to be stimulated by UVB. Treatment with L. pumila extract markedly inhibited the TNF-α production and the expression of COX-2. Decreased collagen synthesis of human fibroblasts by UVB was restored back to normal level after treatment with L. pumila extract. On the other hand, the enhanced MMP-1 expression upon UVB irradiation was down regulated by L. pumila extract in a dose-dependent manner. Furthermore, treatment of normal keratinocytes with L. pumila extract attenuated UVB-induced MMP-9 expression. These results collectively suggest L. pumila extract has tremendous potential as an anti-photoaging cosmetic ingredient.     

Two Kaempferol Glycosides Separated from Camellia Oleifera Meal by High‐Speed Countercurrent Chromatography and Their Possible Application for Antioxidation

Recently, kaempferol and its glycosides have attracted considerable attention owing to their potentially health‐benefitting properties including protection against chronic diseases. Here, a microwave‐assisted extraction (MAE) method was developed for the extraction of total flavonoid glycosides (FG) from Camellia oleifera meal, a major agrifood waste largely generated as a byproduct from the Camellia oil processing industry. Compared with traditional extraction methods, MAE enables more efficient extraction of FG. High‐speed countercurrent chromatography was then applied to separate FG from MAE extract, and two major compounds were successfully separated with purities above 90.0% as determined by HPLC. These two compounds were further identified by UV, FT‐IR, ESI‐MS, ¹H‐NMR, and ¹³C‐NMR as kaempferol 3‐O‐[α‐L‐rhamnopyranosyl‐(1→6)‐β‐D‐glucopyranosyl]‐7‐O‐β‐D‐glucopyranoside and kaempferol 3‐O‐[β‐D‐glucopyranosyl‐(1→4)‐α‐L‐rhamnopyranosyl]‐7‐O‐α‐L‐rhamnopyranoside, which were for the first time separated from C. oleifera meal. The results of antioxidant activity assay demonstrated that both compounds had excellent scavenging activity for DPPH radical, and exhibited protective effects against H₂O₂‐induced oxidative damage of vascular endothelial cells. The findings of this work suggest the possibility of employing C. oleifera meal as an attractive source of health‐promoting compounds, and at the same time facilitate its high‐value reuse and reduction of environmental burden.     

Simultaneous determination of sorbic and benzoic acids in milk products using an optimised microextraction technique followed by gas chromatography

A rapid and reliable method for direct determination of sorbic and benzoic acids in milk products was developed by dispersive liquid–liquid microextraction (DLLME) and gas chromatography with flame ionisation detector (GC-FID). A response surface methodology (RSM) based on a central composite design (CCD) was applied for optimisation of the main variables, such as volume of extraction and dispersive solvents, pH and salt effect. The primary extraction of sorbic and benzoic acids were performed in 8 mL NaOH (0.1 M) in a closed-vessel system. Carrez solutions (potassium hexaferrocyanide and zinc acetate) were used for protein sedimentation. The best simultaneous extraction efficiency was identified using acetone and 1-octanal as dispersive and extraction solvents, respectively. For DLLME, central composite design resulted in the optimised values of microextraction parameters as follows: 475 µL of dispersive and 60 µL of extraction solvents, 2 g NaCl at pH 2.5. Under optimum conditions, the calibration curve was linear over the range 0.1–50 μg mL^?1 and the square of correlation coefficient (R²) was 0.9992 for sorbic acid and 0.9994 for benzoic acid. Relative standard deviation (RSD %) was 6.1% and 3.1% (n = 5) for sorbic and benzoic acids, respectively. Limits of detection were 150 ng g^?1 for sorbic acid and 140 ng g^?1 for benzoic acid and recoveries were 88% and 103.7% respectively. Good reproducibility (RSD %), short extraction time and no matrix interference were advantages of the proposed method which was successfully applied to the determination of sorbic and benzoic acids in milk products.     

Analysis of Flavonoids in <Emphasis Type="Italic">Portulaca oleracea</Emphasis> L. by UV–Vis Spectrophotometry with Comparative Study on Different Extraction Technologies

Portulaca oleracea L. is a traditional edible and medicinal plant in China. Flavonoids are one of the main active ingredients of this plant. Five extraction technologies of flavonoids from P. oleracea L. were investigated and compared, including microwave-assisted extraction, ultrasonic extraction, reflux extraction, Soxhlet extraction, and marinated extraction. The results showed that microwave-assisted extraction was most suitable for the extraction of flavonoids from P. oleracea L. because of its high effect and short extraction time. The found optimum extraction conditions were that the ethanol concentration was 70% (v/v), solid–liquid ratio was 1:50, extracting temperature was 50 °C and irradiation time was 9 min. Quantification was performed by means of UV–Vis spectrophotometry with chromogenic system of NaNO₂–Al (NO₃)₃–NaOH. Under the optimum conditions, the calibration curve for the analyte was linear with the correlation coefficients greater than 0.9999. The average recovery was 102.6%, and its RSD was 1.13%(n = 5). Eight types of P. oleracea L. according to different habits were investigated. The total content of flavonoids was 7.16, 7.10, 9.38, 6.82, 6.78, 11.36, 5.12, and 1.76 mg g⁻¹, respectively.     

Bog Bilberry (Vaccinium uliginosum L.) Extract Reduces Cultured Hep-G2, Caco-2, and 3T3-L1 Cell Viability,Affects Cell Cycle Progression,and Has Variable Effects on Membrane Permeability

ABSTRACT: Bog bilberry (Vaccinium uliginosum L.) is a blue-pigmented edible berry related to bilberry (Vaccinium myrtillus L.) and the common blueberry (Vaccinium corymbosum). The objective of this study was to investigate the effect of a bog bilberry anthocyanin extract (BBAE) on cell growth, membrane permeability, and cell cycle of 2 malignant cancer cell lines, Caco-2 and Hep-G2, and a nonmalignant murine 3T3-L1 cell line. BBAE contained 3 identified anthocyanins. The most abundant anthocyanin was cyanidin-3-glucoside (140.9 ± 2.6 μg/mg of dry weight), followed by malvidin-3-glucoside (10.3 ± 0.3 μg/mg) and malvidin-3-galactoside (8.1 ± 0.4 μg/mg). Hep-G2 LC50 was calculated to be 0.563 ± 0.04 mg/mL, Caco-2 LC50 was 0.390 ± 0.30 mg/mL and 0.214 ± 0.02 mg/mL for 3T3-L1 cells. LDH release, a marker of membrane permeability, was significantly increased in Hep-G2 cells and Caco-2 cells after 48 and 72 h compared to 24 h. The increase was 21% at 48 h and 57% at 72 h in Caco-2 cells and 66% and 139% in Hep-G2 cells compared to 24 h. However, 3T3-L1 cells showed an unexpected significant lower LDH activity (P ≤ 0.05) after 72 h of exposure corresponding to a 21% reduction in LDH release. BBAE treatment increased sub-G1 in all 3 cell lines without influencing cells in the G2/M phase. BBAE treatment reduced the growth and increased the accumulation of sub-G1 cells in 2 malignant and 1 nonmalignant cell line; however, the effect on membrane permeability differs considerably between the malignant and nonmalignant cells and may in part be due to differences in cellular membrane composition.     

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues – a potential cosmetic role

The naturally occurring tetrapeptide acetyl‐N‐Ser‐Asp‐Lys‐Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10^?11–10^?7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10^?5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo‐cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10^?11–10^?7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin‐1, occludin, ZO‐1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two‐fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10^?11–10^?7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.     

J. Cosmet. Sci., 59, 419–430 (September/October 2008)Anti-inflammatory activity of Crinum asiaticum Linne var. japonicum extract and its application as a cosmeceutical ingredient

Synopsis Crinum asiaticum Linne var. japonicum has long been used as a rheumatic remedy, as an anti‐pyretic and as an anti‐ulcer treatment, and for the alleviation of local pain and fever in Korea and Malaysia. In order to investigate the possibility of Crinum asiaticum Linne var. japonicum extract as a cosmetic ingredient, we measured its anti‐inflammatory effect by its inhibition of iNOS (inducible nitric oxide synthase) and the release of PGE2, IL‐6, and IL‐8. We also measured its anti‐allergic effect by its inhibition of beta‐hexosamidase release. An HPLC experiment after extraction with 95% EtOH at pH 3.5 showed that Crinum asiaticum Linne var. japonicum was mainly composed of lycorine (up to 1%), a well‐known immunosuppressor. The content of lycorine varied, depending on the type of plant tissue analysed and the extraction method. In an anti‐inflammatory assay for inhibition of nitric oxide formation on lipopolysaccharide (LPS)‐activated mouse macrophage RAW 264.7 cells, the ethanol extract of Crinum asiaticum showed an inhibitory activity of NO production in a dose‐dependent manner (IC50 = 58.5 μg ml^‐1). Additional study by RT‐PCR demonstrated that the extract of Crinum asiaticum significantly suppressed the expression of the iNOS gene. Moreover, the extract of Crinum asiaticum did not show any cytotoxicity, but did show a cell proliferation effect against LPS (a 10–60% increase in cell viability). In an assay to determine inhibition of the H2O2‐activated release of PGE2, IL‐6, and IL‐8 in human normal fibroblast cell lines, the release of PGE2 and IL‐6 was almost completely inhibited above concentrations of 0.05% and 1%, respectively. Moreover, the release of IL‐8 was completely inhibited over the entire range of concentration (> 0.0025%). In order to investigate the skin‐sensitizing potentials of the extract of Crinum asiaticum, a human clinical test was performed after repeated epicutaneous 48‐h applicaticons under an occlusive patch (RIPT). The repeated and single cutaneous applications of Crinum asiaticum Linne var. japonicum extract under the occlusive patch did not provoke any cumulative irritation and sensitization reactions. The result showed that the extract of Crinum asiaticum Linne var. japonicum has a sufficient anti‐inflammatory effect. Therefore, Crinum asiaticum Linne var. japonicum extract may be useful for development as an ingredient in cosmetic products.     

Shelf life of alginate beads containing lactobacilli and bifidobacteria: characterisation of microspheres containing Lactobacillus delbrueckii subsp. bulgaricus

Capsules, containing different microorganisms (Lactobacillus plantarum, L. casei, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. reuteri, L. rhamnosus, Bifidobacterium breve and B. animalis subsp. lactis), were produced through a simple extrusion protocol and dipped in a CaCl₂ solution (0.5% or 8%). Beads were characterised, to assess the encapsulation yield (EY) and the shelf life at 4 °C (defined as the time to retain inside a cell count of 6 log CFU g^?1); then, L. delbrueckii subsp. bulgaricus was used as target for system optimisation, through the study of the kinetic of cell release, as a function of the concentration of CaCl₂ solution, agitation (150 rpm) and recycling. The EY was 50% or higher, and the shelf life was at least 30 days. Concerning beads containing L. delbrueckii subsp. bulgaricus, the release of cells from capsules followed a biphasic (diauxic) trend and the microsphere could be used successfully for two times, as only after third reuse, the amount of release cells decreased.     

Optimization of microwave-assisted extraction of anthocyanins in red raspberries and identification of anthocyanin of extracts using high-performance liquid chromatography – mass spectrometry

Anthocyanins (Acys) are naturally occurring compounds that impart color to fruit, vegetables, and plants. The extraction of Acys from red raspberry (Rubus idaeus L. var. Heritage) by microwave-assisted process (MAP) was studied. A central composite rotate design (CCRD) was used to obtain the optimal conditions of microwave-assisted extraction (MAE), and the effects of operating conditions such as the ratio of solvents to materials, microwave power and extraction time on the extraction yield of Acys were studied through response surface methodology (RSM). The optimized conditions of MAE were ratio of solvents to materials 4:1 (ml/g), extraction time 12 min, and microwave power 366 W. Under these conditions 43.42 mg of Acys from 100 g of fresh fruits (T _Acy, expressed as cyanidin-3-glucoside), approximately 98.33% of the total red pigments, could be obtained by MAE. The Acys compositions of extracts were identified by high-performance liquid chromatography – mass spectrometry (HPLC-MS), 12 kinds of Acys had been detected and 8 kinds of Acys were characterized. Result indicated that cyanidin-3-sophoroside, cyanidin-3-(2 ^G -glucosylrutinoside), cyanidin-3-sambubioside, cyanidin-3-rutinoside, cyanidin-3-xylosylrutinoside, cyanidin-3-(2 ^G -glucosylrutinoside), and cyanidin-3-rutinoside were main components in extracts. In addition, in comparison with the conventional solvent extraction, MAE is more efficient and rapid to extract Acys from red raspberry, due to the strong disruption of fruit tissue structure under microwave irradiation, which had been observed with the scanning electron microscopepy (SEM). However, the Acys compositions in extracts by both the methods were similar, which were investigated using HPLC profile.     

Synergistic cytotoxicity induced by α-solanine and α-chaconine

α-Solanine and α-chaconine are well-known potato toxins, but the mechanism of the synergistic cytotoxic effect of these alkaloids has been little clarified. This study confirmed their synergistic cytotoxic effects on C6 rat glioma cells by three different cell viability tests, namely WST-1 (water-soluble tetrazolium) assay sensitive to intracellular NADH concentration, menadione-catalysed chemiluminescent assay depending on both NAD(P)H concentration and NAD(P)H:quinone reductase activity, and LDH (lactate dehydrogenase) assay sensitive to the release of LDH from damaged cells. The maximum cytotoxic effect was observed at a ratio of 1:1 between α-solanine and α-chaconine at micromolar concentrations. The cytotoxic effects of these alkaloids were observed immediately after incubation and were constant after 30 min, suggesting that rapid damage of plasma membrane causes the lethal disorder of metabolism.