Volume 44, Issue 3

Objective Eucalyptus camaldulensis is recognized to be one of the major introduced eucalypts, the plant extracts possess a wide range of phytochemicals, which are accountable for its antioxidant and pharmacological activities. The extraction efficiency of such chemical compounds is influenced by the applied extraction conditions. To test the effect of solvent type on the yield of phenolics and antioxidants from E. camaldulensis , seeking for an optimization of the extraction process. Method Dry powders of four plant organs were extracted by some organic solvents besides water. The total phenolic content was detected in the crude extracts of leaf, bud, empty capsule and seed, and was further characterized by GC-MS. Results Acetone-water mixtures yielded high amounts of phenolic compounds with parallel antioxidant activities, correlation coefficients were significant (0.696–0.998). Bud and capsule are first accredited for their antioxidant potentials. The GC-MS analysis revealed the abundance of most detected phenolic constituents in the plant seed. Discussion Acetone-water is a successful and appropriate mixture for the extraction of phenolic compounds from E. camaldulensis . It could give better yields and enhance the biological activities of its corresponding extracts. Conclusion The accurate choice of an extraction solvent has a great influence on the yields of phenolics.

Background Universal demand for cheese keeps the search for appropriate enzymes from plants mimicking animal rennet action in scientific focus. Objective To associate distribution of milk clotting potential and profile of whole/κ-casein hydrolysis by Calotropis gigantea stem, leaf and respective calli crude enzymes (CE). Materials and methods Milk clotting activity and index were assayed for CE. Caseinolytic activity (CA) was evaluated spectrophotometrically. 0.5 CA units of CE and Enzeco ® were used for studying whole/κ-casein hydrolysis pattern by Tricine SDS-PAGE. Inhibition studies were performed for enzyme characterisation. Results Traditionally propagated (TP) stem and its callus CE exhibited high specific milk clotting activity (1297.30±0.2 U/mg of protein and 926.74±44.13 U/mg of protein, respectively) and milk clotting index (103.562±1.162 and 79.365±4.93, respectively). Comparison of whole casein hydrolytic pattern by 0.5 CA units of CE revealed closer resemblance between leaf callus and Enzeco ® . However, κ-casein specificity analysis revealed TP leaf to be closely mimicking the performance by Enzeco ® . Conclusion Study suggests CE from TP leaf to be a potential vegetable coagulant to work as an effective and low-cost milk clotting mediator in cheese industry.

Background Pollen, mold spores, bacteria and viruses are the main biological substances in the atmosphere causing allergic symptoms and disease. Distinguishing pollen and spores is quite time consuming and requires a trained expert. There is a different approach to identification of these substances such as microscopic analysis. However, DNA based identification of these is becoming popular recently. Objective We evaluated the correlation between the quantity of DNA, which was amplified using trn T-F cpDNA specific primers in samples obtained from a high volume air sampler (HVAS), and concentration of Poaceae pollen collected with a Burkard trap. Materials and methods Here, we present a method for identifying and quantifying airborne Poaceae pollen using a single step polymerase chain reaction (PCR) technique. Forty daily air samples were collected by HVAS. The method was optimised using two different methods (M1 and M2) and the trn T-F cpDNA region was amplified using a Poaceae specific primer pair. The correlation between the quantity of DNA and pollen concentration was tested using R statistical programming language. Results Although a significant correlation was obtained between the M1 and M2 methods (R 2 =0.655, p<0.01), the M2 method was more correlated with pollen concentration. The correlation between pollen and DNA content changed due to episodes that were observed during the pollen season. DNA concentrations from the PCR data were significantly correlated with pollen concentrations determined by light microscopy (R 2 =0.767, p<0.01) in episode II using the M2 method and during the entire season (R 2 =0.469, p<0.01) using M2. Conclusions The M2 method correctly identified Poaceae pollen in mixed air samples from Zonguldak Province. The non-coding trn T-F cpDNA region was used for the first time in aerobiological samples to identify Poaceae pollen. Use of this method that does not require DNA extraction may be a crucial step for real-time pollen monitoring devices to be developed in the future. The correlation strength between pollen and amplified DNA content could be improved using a sampler that has a lower absorption rate, and a more sensitive technique, such as qPCR.

Objective Targeting the protein of interest to a particular tissue to achieve high-level expression is an important strategy to increase expression efficiency. The use of the plant seed oil body as a bioreactor can not only increase the amount of target protein, but also reduce the cost of downstream processing. Methods VEGF 165 was expressed in Arabidopsis thaliana seeds via oilbody fusion technology. The pKO-VEGF 165 vector was construted and transformed into A. thaliana seeds. T3 transgenic seeds was detected by SDS-PAGE and western blot methods. The cell activity was tested by MTT methods. Result The phaseolin promoter was used to drive seed-specific expression of the VEGF 165 gene in transgenic A. thaliana . The coding region of VEGF 165 was fused to the Arabidopsis oleosin sequence to target the protein to the oil bodies in the seeds of transgenic plants. The T-DNA region of recombinant plasmid pKO-VEGF 165 was shifted to A. thaliana seeds via the floral-dip method. Protein was analyzed by electrophoresis and protein hybridization analyses. Finally, MTT assays showed that the oleosin-VEGF 165 fusion protein played a part in the proliferation of HUVEC cells in vitro. Conclusion Oleosin-VEGF 165 was successfully expressed and it had stimulated HUVEC cell proliferation activity.

Background Dengue fever has emerged as a serious threat in Pakistan in the last few years with high morbidity rates and substantial mortality. In the present study, NS2B/NS3 protease from four dengue virus (DENV) serotypes have been targeted using 2350 phytochemicals from various medicinal plants. Material and methods The phytochemicals were subjected to docking against NS2B/NS3 proteases using AutoDock Vina focusing the binding site, and the binding energies were determined to screen the effectively docked phytochemicals. Pharmacological properties were also analyzed for all the phytochemicals using PreADMET web server. Results Binding affinities ranged from −4.0 to –9.8 kcal/mol and a threshold of −9.0 kcal/mol was applied for screening compounds. A total of 18 phytochemicals are screened for passing all evaluation criteria of a drug in which three were for DENV1-NS2B/NS3, five for DENV2-NS2B/NS3, six for DENV3-NS2B/NS3 and four for DENV4-NS2B/NS3. Erycristagallin and Osajin from Erythrina variegate , PapraineA from Fumaria indica and Aloe-Emodin from Aloe vera are the most potent inhibitors of NS2B/NS3 protease from DENV1, DENV2, DENV3 and DENV4, having binding affinities of −9.6 kcal/mol, −9.6 kcal/mol, −9.6 kcal/mol and −9.2 kcal/mol, respectively. Conclusion The effective drug-like properties of all 18 phytochemicals demonstrate the inhibition potential against dengue virus replication in human beings.

Objectives To determine of antioxidant, cytotoxic and tyrosinase inhibitory (TI) activities, and phenolic profiles with a new high performance liquid chromatography (HPLC) method of green, white and black teas. Methods Antioxidant activities of the teas were examined by means of scavenging of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and cupric reducing antioxidant capacity (CUPRAC). The phenolic contents were investigated by means of HPLC. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxic potential of tea extracts in cancer and normal cell lines. TI activity was investigated against diphenolase (DOPA), using kojic acid as a positive control. Results The green tea extract exhibited stronger antioxidant than white and black tea extracts. The green tea contained syringaldehyde, p -coumaric acid, benzoic acid, and quercetin. The white tea extract had the highest tyrosinase inhibitor activity. The extracts exhibited higher cytotoxic potential toward cancer cells than normal cells. The methanol extract of green tea had the highest cytotoxic potential, while the water extracts were less toxic. Conclusion The green, white, and black teas can be regarded as an important in terms of rich antioxidant, tyrosinase inhibitor, and cytotoxic activities in both the pharmaceutical and food industries.

Objectives Rhodomyrtus tomentosa ( Aiton. ) Hassk. ( R. tomentosa ) is rich in nutrients and has multiple pharmacological applications. Anthocyanins confer color to the flowers and berries of R. tomentosa and provide protection against photodamage. The dihydroflavonol 4-reductase gene ( DFR ) and phenylalanine ammonialyase gene ( PAL ) are crucial for anthocyanin synthesis. Methods DFR and PAL transcript levels and anthocyanin content in the pigmented organs of R. tomentosa were investigated through qRT-PCR analysis and spectrophotometry, respectively. The glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) gene was selected as the reference gene for the normalization of DFR and PAL transcript levels. Results Transcript levels of DFR and PAL were higher in organs with vigorous metabolism than those in senescent organs. DFR and PAL transcript levels were up-regulated during the initial and middle-maturity periods of fruit. These expression patterns are consistent with fruit color development. The highest transcript levels of PAL and DFR were observed during the middle-maturity period or the red-fruit period. Conclusion During the late maturity period of R. tomentosa fruit, the transcript levels of the two genes were down-regulated even though anthocyanins were continuously accumulated, which was different from the accumulation of anthocyanins in some late mature fruits.

Background Aboveground parts and bulbs of Ornithogalum species are consumed as food and used in traditional medicine in worldwide. Objective It is aimed to report the antioxidant capacity and phenolic compounds content of Ornithogalum sigmoideum , Ornithogalum orthophyllum and Ornithogalum oligophyllum for the first time. Materials and methods Antioxidant activity of the crude methanol extracts of the aerial parts and the bulbs of the species were determined with 1,1-diphenyl-2-picryl-hydrazyl, superoxide radical scavenging, ferrous ion-chelating effect, phosphomolybdenum-reducing antioxidant power and ferric-reducing antioxidant power assay. The ethylacetate, diethylether and water subextracts from leaf and flower were analyzed to quantify selected phenolic compounds by HPLC-UV. Results Among the six extracts, the methanol extract of the aerial parts of O. orthophyllum contained the highest amount of phenolic compounds (GAE, 11.0 mg/g extract). The aerial parts of O. orthophyllum showed higher DPPH and SOD activities than the other extracts with the SC 50 values of 0.39±0.05 mg/mL and 0.44±0.08 mg/mL, respectively. Protocatechuic acid, p -hydroxybenzoic acid, vanillic acid and p -coumaric acid were the most abundant compounds at all the subextracts. Conclusion The antioxidant activity is found to be in accordance with the levels of phenolic content in the extracts.

Background The present study reports the optimization of the biotechnological conditions in order to improve the lactic acid fermentation process, in a rye flour medium by using Lactobacillus paracasei ssp. paracasei strain, coded MIUG BL6, as a starter culture. Materials and methods A sequential statistical methodology, comprised of Plackett-Burman experimental design, central composite design and response surface methodology, was applied to enhance the lactic acid fermentation productivity. Results and discussion Among the tested parameters, the milk powder and KH 2 PO 4 concentration and also the time of fermentation were identified as the most significant variables that influence the fermentation process. The optimum levels of these significant parameters were determined as follows: 4.21% milk powder, 0.30% KH 2 PO 4 and 48 h of fermentation that induced an increase of 1.6 fold of the acidity, compared to the fermentation conducted under the non-optimized conditions. Also, under these optimized fermentation conditions, a good rate of cell multiplication of 10.19 log N/N 0 was achieved.

Objective The objective of this study was to characterize flours and syrups, obtained from pods of carob ( Ceratonia siliqua L.) and honey locust ( Gleditsia triacanthos ). Method Flours and syrups, produced by carob and honey locust were analyzed for moisture, ash, protein content, dietary fibers, minerals composition, total phenolic content, as well as antibacterial and antioxidant activity. Results and discussion Carob flour contained high amounts of protein (22.56%) and dietary fibres (28.17%), respectively. Dietary fibers in honey locust flour (33.12%) were higher than that of carob flour (28.17%). The total phenolic content of carob flour (4.53±0.08) was lower than this of honey locust (25.31±0.06) (mg gallic acid equivalent [GAE]/g dry weight). Gleditsia triacanthos flour showed higher antioxidant potential – from 127.52±2.43 to 540.28±2.47 μM TE/g dw. Carob syrup in an amount of 0.15 cm 3 demonstrated pronounced antibacterial activity against Listeria monocytogenes , Escherichia coli , Salmonella enterica and Staphylococcus aureus , respectively. Conclusion The current study demonstrated that flour and syrup, obtained from carob ( C. siliqua L.) and honey locust ( G. triacanthos ) pods presented products rich of protein and dietary fiber (both above 20%), good sources of antioxidants, especially poliphenolic compounds and minerals (Mg, Fe and Zn).

Background Biological control of pathogenic fungi is a possible alternate to the chemical control, which is harmful to humans and environment. Soil-borne Bacillus strains can be potential biocontrol agents and a source of lytic enzymes. Aim This study aimed to examine biocontrol potential and lytic enzyme activities of a soil isolate Bacillus subtilis Ege-B-1.19. Materials and methods Strain was identified by biochemical and 16S rRNA gene analysis and its biocontrol activity was investigated against Aspergillus niger EGE-K-213, Aspergillus foetidus EGE-K-211, Aspergillus ochraceus EGE-K-217, Fusarium solani KCTC6328, Rhizoctonia solani KACC40111 and Colletotrichum gloeosporioides KACC40689. Chitinase, chitosanase, N-acetyl-β-hexosaminidase and protease activities of B. subtilis Ege-B-1.19 were also determined. Chitosanase was purified using Sephadex G-150 column and its molecular weight was determined by SDS-PAGE. Chitooligosaccharides production using chitosanase was carried out and analysed by TLC and HPLC. Results Results depicted that B. subtilis Ege-B-1.19 has shown inhibitory effects against all the test fungi. Chitinase, chitosanase, N-acetyl-β-hexosaminidase and protease activities were determined as 2.7 U mL −1 , 7.2 U mL −1 , 6.2 U mL −1 and 12.2 U mL −1 , respectively. Molecular weight of purified chitosanase was 44 kDa. Chitosanase hydrolysed chitosan to glucosamine (GlcN), dimers (GlcN) 2 and trimers (GlcN) 3 . Conclusion Bacillus subtilis Ege-B-1.19 can be anticipated as useful biocontrol agent and its chitosanase can be utilized for enzymatic synthesis of chitooligosaccharides.

Background Indian fish industry produces a staggering amount of waste every year part of which is dumped as landfill leading to pollution. Objective This study aims to extract collagen from fish skin, a major component of this waste, hydrolyze it and use the hydrolysate as emulsifiers to increase the shelf life of food products. Materials and methods Collagen was purified from the skin of Catfish, Mullet and Indian Salmon were characterized through infrared and diffraction studies. The samples were hydrolyzed enzymatically. The hydrolysates were assayed for emulsifying, foam stabilizing and hemolytic properties. Results The samples displayed an electrophoretic and infrared spectral profile corresponding to that of collagen type I. X-ray diffraction patterns revealed the presence of triple helix and traces of phosphate. The hydrolysates (6 kDa) exhibited the highest emulsion at 2.74±0.20 m 2 /g (for 57.00±0.50 min) and foam stability in the range of 75–78%, respectively. The hydrolysates were able to stabilize drug and food formulations successfully and two of the samples exhibited less than 3% toxicity as determined through hemolytic assay. Conclusion Collagen hydrolysates from fish skin, a common fish industry waste, were found to be bioactive and non-toxic making them suitable replacements for synthetic emulsifiers.

Background Poly-β-hydroxybutyrate (PHB) is the most important and versatile class of biodegradable polymers used successfully in the medical, agricultural and industrial field. Idea is to find the novel isolate for degradation of biodegradable plastics that can enhance the bioremediation. Materials and methods Thirty-one PHB and PHB depolymerase enzyme producing isolates out of 80 mesophilic bacteria from Lucknow region were further screened for PHB degradation capability by secreting extracellular PHB depolymerase enzyme in minimal salt media supplemented with PHB (0.15%). Various biodegradable plastic films were tested by soil burial method for weight loss determination. Result 37.3% weight loss has been observed in PHB films when buried under the soil for 45 days in the presence of a novel PHB degrader identified as Paenibacillus alvei PHB28 by 16S rRNA sequencing (GenBank accession number KX886342). These Gram-negative, spore-forming, thermotolerant bacteria produce maximum PHB depolymerase (5.03 U/mL) at 45°C, pH 8.0, with 0.15% substrate concentration when incubated for 96 h with starch (0.1%) and yeast extract (0.01%) as an additional nutrient supplements. Conclusion To the best of our knowledge this is the first report of PHB depolymerase production by P. alvei PHB28 which may contribute successfully to combat plastic pollution and to sustain the green environment.

Background Deer antler is the only regenerative organ in mammals, the regeneration of antler is not only the regeneration of bone tissue, but also accompanied by the regeneration of nerves, blood vessels and so on. The purpose of the current study was to explore the effect of miRNA-15a and miRNA-15b on the regulation of sika deer vascular endothelial growth factor receptor (VEGFR) during rapid antler growth. Materials and methods The VEGFR 3′-UTR was analyzed by bioinformatics software to identify the highly matched miRNAs. After transfected with miRNA mimics, the expression of selected miRNAs were measured by RT-qPCR and the relative expression level of VEGFR protein was detected by Western Blot. Dual-luciferase activity assay was used to determine the target relationship between VEGFR and miRNAs. The cartilage cell proliferation and telomerase activity were measured by MTT kit and TRAP assay, respectively. Results The VEGFR 3′-UTR contains a binding site for miRNA-15a and miRNA-15b. Over-expression of miRNA-15a and miRNA-15b, which significantly reduced the expression level of VEGFR protein, inhibited the proliferation of cartilage cells, and decreased the telomerase activity of cartilage cells in vitro. Conclusion miRNA-15a and miRNA-15b represent novel regulatory factors of VEGFR expression in deer antler.

Background To investigate the interaction of pendimethalin (PM), a commonly used herbicide, with various mammalian serum albumins. Methods The interactions of PM with serum albumins of bovine (BSA), sheep (SSA), porcine (PSA) and rabbit (RbSA) were studied using fluorescence quenching titration and site marker displacement experiments. Results A comparison of the PM-induced quenching of the fluorescence of these albumins with that published for human serum albumin (HSA) showed similarity between BSA and HSA. The PM binding affinity of these albumins was found to follow the order: SSA>BSA>RbSA>PSA. Warfarin (WFN) displacement results also suggested similar displacing action of PM on WFN-BSA complex, when compared to the published results on WFN-HSA complex. Conclusion The results suggested close similarity between BSA and HSA in terms of PM binding characteristics and hence bovine can be selected as a suitable animal model for further toxicological studies of PM.

Background CCl 4 is a widely used xenobiotic for the purpose of causing liver damage in experimental studies. In this study, we aimed to reveal the effects of crocin on liver injury caused by CCl 4 via free radical scavenging properties. Materials and methods Animals were divided into five groups of 10: control; corn oil; crocin; CCl 4 ; CCl 4  + crocin. Tissue samples were carefully removed and separated for biochemical and histological investigations. Results CCl 4 administration led to significant increases in MDA, SOD, CAT and TOS in liver tissue, and AST, ALT and ALP levels in plasma (p < 0.05). In addition, CCl 4 caused significant decreases in GSH and TAS (p < 0.05). When animals were treated with crocin, high MDA, SOD, CAT, TOS levels, and AST and ALP activities decreased and GSH and TAS levels increased. Control group exhibited normal histological appearance; however extensive necrosis areas were detected in the CCl 4 group. In the CCl 4  + crocin group, pathological changes were markedly decreased and the appearance of liver tissue was almost similar to the control groups. Conclusion Our results showed that crocin suppresses oxidative stress with antioxidant properties and has a protective effect on tissue damage caused by CCl 4 .

Background and objective Ethyl glucuronide (EtG) and ethyl sulfate (EtS), minor metabolites of ethanol, aid to forensic scientist and clinicians to determine recent ethanol uptake when ethanol elimination is occurred. Present study aimed to show (a) kinetics of EtG and EtS in blood and urine after 0.5 g/kg ethanol intake (b) peak concentrations and time of disappearance in Turkish people. Material and methods Seventeen (10 male, 7 female) healthy volunteers participated in the study. Blood and urine samples were obtained during 48 h after consumption and analyzed in LC-MS/MS. Results and conclusion Blood peak concentrations of EtG and EtS were 0.13–0.389 mg/L and 0.211–0.5 mg/L, urine peak concentrations were 6.89–30.42 mg/L and 10.5–58.17 mg/L, respectively. There was no EtG and EtS in all samples 24 h later in blood and 48 h later in urine. Correlation was found between EtG and EtS concentrations in blood. Most of obtained data are similar to literature, except EtS dominancy to EtG in urine and blood.

Background Essential oils are chemical products produced by odoriferous glands from a variety of plants. These essential oil have many health benefits i.e. antiseptic, anti-inflammatory and antimicrobial activities. So due to these medicinal properties present study was designed to analyze essential oil of Thymus vulgaris and Mentha arvensis for their chemical composition and biological activities. Materials and methods Essential oil from these plants were extracted by hydrodistillation method, and analyzed by GC-MS. To test the microbial activity of these oil disk diffusion method and micro wells method were used. For free radical scavenging DPPH assay was used. However total phenolic content was measured by colorimetric method. Results The GC-MS analysis of T. vulgaris oil showed the presence of 47 chemical compounds among which thymol, terpinene, p-cymene and carvacrol were major. However essential oil of M. arvensis showed the presence of 28 constituents, among which the Menthone, Menthol, Isomenthone, Eucalyptol, neo-Menthol, cis-Piperitone oxide, Linalool, Thymol, Limonene, and α-Phellandrene were major. Essential oil from both these plant tested for antimicrobial activity showed that the T. vulgaris oil was effective against seven bacterial strains and the essential oil of M. arvensis was effective against six bacterial strain. The antioxidant activity of both samples by DPPH assay which showed positive result. Conclusion As both species showed the presence of active components, positive microbial activities, and antioxidant activity so, research should be carried on for further biological activities of these oil for betterment of living beings.

Background Aim of the study was to immobilize the α-amylase produced earlier from the mesophilic fungus Rhizoctonia solani AG-4 strain ZB-34 by solid-state fermentation and investigate the suitability of immobilized enzymes for some industries. Materials and methods A novel α-amylase from R. solani AG-4 strain ZB-34 was immobilized in chitosan by covalent binding and Ca-alginate by entrapment. Results The efficiency of chitosan and Ca-alginate immobilization was 67.9% and 59.6%, respectively. The immobilized enzymes showed the highest activity in the presence of starch. Optimum values for chitosan and Ca-alginate immobilized enzymes were pH 4.50 and 40°C and pH 5.50 and 60°C, respectively. It was found that immobilized enzymes were highly stable in terms of thermal and pH stabilities. When the chitosan immobilized enzyme was used with detergents, chocolate stains on dirty laundry was better cleaned. Chitosan immobilized R. solani AG-4 strain ZB-34 α-amylase was found to have a higher desizing effect at 40°C in tap water. As a result of Ca-alginate immobilization, the enzyme clarified apple juice more than the free enzyme. Conclusion The results showed that immobilized enzymes might have potential applications in industry. This is the first report immobilizing an α-amylase produced from the fungus R. solani .