Search results for: HPLC method

Authors: Parvaneh Hajeb, Shahram Shakibazadeh, Md. Zaidul Islam Sarker

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High-quality fish oil for human consumption requires low levels of toxic elements. The aim of this study was to develop a method to extract oil from fish wastes with the least toxic elements contamination. Supercritical fluid extraction (SFE) was applied to detoxify fish oils from toxic elements. The SFE unit used consisted of an intelligent HPLC pump equipped with a cooling jacket to deliver CO2. The freeze-dried fish waste sample was extracted by heating in a column oven. Under supercritical conditions, the oil dissolved in CO2 was separated from the supercritical phase using pressure reduction. The SFE parameters (pressure, temperature, CO2 flow rate, and extraction time) were optimized using response surface methodology (RSM) to extract the highest levels of toxic elements. The results showed that toxic elements in fish oil can be reduced using supercritical CO2 at optimum pressure 40 MPa, temperature 61 ºC, CO2 flow rate 3.8 MPa, and extraction time 4.25 hr. There were significant reductions in the mercury (98.2%), cadmium (98.9%), arsenic (96%), and lead contents (99.2%) of the fish oil. The fish oil extracted using this method contained elements at levels that were much lower than the accepted limits of 0.1 μg/g. The reduction of toxic elements using the SFE method was more efficient than that of the conventional methods due to the high selectivity of supercritical CO2 for non-polar compounds.

Keywords: food safety, toxic elements, fish oil, supercritical carbon dioxide

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Authors: Zahid Iqbal, Ijaz Javed, Riaz Hussain, Ibadullah Jan, Amir Ali Khan

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This study was conducted to investigate the disposition kinetics of ciprofloxacin and calculate its optimal dosage in Pakistani sheep of Lohi breed. Injectable preparation of ciprofloxacin was given intramuscularly to eight sheep at a dose of 5 mg/Kg. Before administration of drug blood sample was drawn from each animal. Post drug administration, blood samples were also drawn at various predetermined time periods. Drug concentration in the blood samples was assessed through high performance liquid chromatograph (HPLC). Data were best described by two compartment open model and different pharmacokinetic (PK) parameters were calculated. Cmax of 1.97 ± 0.15 µg/ml was reached at Tmax of 0.88 ± 0.09 hours. Half life of absorption (t1/2 abs) was observed to be 0.63 ± 0.16 hours while t1/2 α (distribution half life) and t1/2 ß (elimination half life) were found to be 0.46 ± 0.05 and 2.93 ± 0.45 hours, respectively. Vd (apparent volume of distribution) was calculated as 2.89 ± 0.30 L/kg while AUC (area under the curve) was 7.19 ± 0.38 µg.hr/mL and CL (total body clearance) was 0.75 ± 0.04 L/hr/kg. Using these parameters, an optimal intramuscular dosage of ciprofloxacin in adult Lohi sheep was calculated as 21.43 mg/kg, advised to be repeated after 24 hours. From this, we came to the conclusion that calculated dose was much higher than the dose advised by the foreign manufacturer and to avoid antimicrobial resistance, it is advised that this locally investigated dosage regimen should be strictly followed in local sheep.

Keywords: pharmacokinetics, dosage regimen, ciprofloxacin, HPLC, sheep

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Authors: A. M. Tarola, R. Preti, A. M. Girelli, P. Campana

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Safeguarding, studying and enhancing biodiversity play an important and indispensable role in re-launching agriculture. The ancient local varieties are therefore a precious resource for genetic and health improvement. In order to protect biodiversity through the recovery and valorization of autochthonous varieties, in this study we analyzed 12 samples of four ancient apple cultivars representative of Friuli Venezia Giulia, selected by local farmers who work on a project for the recovery of ancient apple cultivars. The aim of this study is to evaluate the polyphenolic profile and the antioxidant capacity that characterize the organoleptic and functional qualities of this fruit species, besides having beneficial properties for health. In particular, for each variety, the following compounds were analyzed, both in the skins and in the pulp: gallic acid, catechin, chlorogenic acid, epicatechin, caffeic acid, coumaric acid, ferulic acid, rutin, phlorizin, phloretin and quercetin to highlight any differences in the edible parts of the apple. The analysis of individual phenolic compounds was performed by High Performance Liquid Chromatography (HPLC) coupled with a diode array UV detector (DAD), the antioxidant capacity was estimated using an in vitro essay based on a Free Radical Scavenging Method and the total phenolic compounds was determined using the Folin-Ciocalteau method. From the results, it is evident that the catechins are the most present polyphenols, reaching a value of 140-200 μg/g in the pulp and of 400-500 μg/g in the skin, with the prevalence of epicatechin. Catechins and phlorizin, a dihydrohalcone typical of apples, are always contained in larger quantities in the peel. Total phenolic compounds content was positively correlated with antioxidant activity in apple pulp (r² = 0,850) and peel (r² = 0,820). Comparing the results, differences between the varieties analyzed and between the edible parts (pulp and peel) of the apple were highlighted. In particular, apple peel is richer in polyphenolic compounds than pulp and flavonols are exclusively present in the peel. In conclusion, polyphenols, being antioxidant substances, have confirmed the benefits of fruit in the diet, especially as a prevention and treatment for degenerative diseases. They demonstrated to be also a good marker for the characterization of different apple cultivars. The importance of protecting biodiversity in agriculture was also highlighted through the exploitation of native products and ancient varieties of apples now forgotten.

Keywords: apple, biodiversity, polyphenols, antioxidant activity, HPLC-DAD, characterization

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Authors: Nematollah Gheibi, Nader Divan Khosroshahi, Mahdi Mohammadi Ghanbarlou

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Introduction: Nowadays, the phenomenon of bacterial resistance is one of the most important challenge of the health community in the world. Propolis is most important production of bee colonies that collected from of various plants. So far, a lot of investigations carried out about its antibacterial effects. Material and methods: Thirty gram of propolis prepared as ethanolic extract and after different process of purification, 7.5 gr of its pure form were obtained. Propolis compounds identification was performed by TLC and VLC methods. The HPLC spectrum obtaining from propolis ethanolic extract was compared with some purified standard phenolic and flavonoid substances. Antibacterial effects of ethanol extract of purified propolis were evaluated on two strains of Staphylococcus aureus and Pseudomonas aeruginosa and their MIC was determined by the microdillution assay. Results: Ethanolic propolis extraction analyzed by TLC were resulted to confirm several phenolic and flavonoid compounds in this extract and some of the confirmed by HPLC technique. Minimum inhibitory concentration (MIC) for standard Staphylococcus aureus (ATCC25923) and Pseudomonas aeruginosa (ATCC27853) strains were obtained 2.5 mg/ml and 50 mg/ml respectively. Conclusion: Bee Propolis is a mix organic compound that has a lot of beneficial effects such as anti-bacterial that emphasized in this investigation. It is proposed as a rich source of natural phenolic and flavonoids compounds in designing of new biological resources for hygienic and medical applications.

Keywords: propolis, Staphylococcus aureus, Pseudomonas aeruginosa, antibacterial

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Authors: Samiyah Tasleem, Muhammad Abdul Haq, Syed Baqir Shyum Naqvi, Muhammad Abid Husnain, Sajjad Haider Naqvi

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The objective of the present study was: a) to investigate the antimicrobial activity of honey samples of different floral sources of Pakistan, b) to recover the phenolic acids in them as a possible contributing factor of antimicrobial activity. Six honey samples from different floral sources, namely: Trachysperm copticum, Acacia species, Helianthus annuus, Carissa opaca, Zizyphus and Magnifera indica were used. The antimicrobial activity was investigated by the disc diffusion method against eight freshly isolated clinical isolates (Staphylococci aureus, Staphylococci epidermidis, Streptococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumonia, Escherichia coli, Proteus vulgaris and Candida albicans). Antimicrobial activity of honey was compared with five commercial antibiotics, namely: doxycycline (DO-30ug/mL), oxytetracycline (OT-30ug/mL), clarithromycin (CLR–15ug/mL), moxifloxacin (MXF-5ug/mL) and nystatin (NT – 100 UT). The fractions responsible for antimicrobial activity were extracted using ethyl acetate. Solid phase extraction (SPE) was used to recover the phenolic acids of honey samples. Identification was carried out via High-Performance Liquid Chromatography (HPLC). The results indicated that antimicrobial activity was present in all honey samples and found comparable to the antibiotics used in the study. In the microbiological assay, the ethyl acetate honey extract was found to exhibit a very promising antimicrobial activity against all the microorganisms tested, indicating the existence of phenolic compounds. Six phenolic acids, namely: gallic, caffeic, ferulic, vanillic, benzoic and cinnamic acids were identified besides some unknown substance by HPLC. In conclusion, Pakistani honey samples showed a broad spectrum antibacterial and promising antifungal activity. Identification of six different phenolic acids showed that Pakistani honey samples are rich sources of phenolic compounds that could be the contributing factor of antimicrobial activity.

Keywords: Pakistani honey, antimicrobial activity, Phenolic acids eg.gallic, caffeic, ferulic, vanillic, benzoic and cinnamic acids

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Authors: Namsoo Kim, So-Hee Son, Jin-Soo Maeng, Yong-Jin Cho, Chong-Tai Kim

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It has been reported that some enzymes such as trypsin and Alcalase 2.4L are tolerant to a medium high pressure of 300 MPa and preparation of protein hydrolyzates under 300 MPa was advantageous with regard to hydrolysis rate and thus production yield compared with the counterpart under ambient pressure.1,2) In this study, nine fish comprising halibut, soft shell clam and carp were hydrolyzed using Flavourzyme 500MG only, and the combination of Flavourzyme 500 mg, Alcalase 2.4 L, Marugoto E, and Protamex under 300 MPa. Then, the effects of single and multiple protease treatments were determined with respect to contents of soluble solid (SS) and soluble nitrogen, sensory attributes, electrophoretic profiles, and HPLC peak patterns of the fish hydrolyzates (FHs) from various species. The contents of SS of the FHs were quite species-specific and the hydrolyzates of halibut showed the highest SS contents. At this point, multiple protease treatment increased SS content conspicuously in all fish tested. The contents of total soluble nitrogen and TCA-soluble nitrogen were well correlated with those of SS irrespective of fish species and methods of enzyme treatment. Also, it was noticed that multiple protease treatment improved sensory attributes of the FHs considerably. Electropherograms of the FHs showed fast migrating peptide bands that had the molecular masses mostly lower than 1 kDa and this was confirmed by peptide patterns from HPLC analysis for some FHs that had good sensory quality.

Keywords: production, fish hydrolyzates, protease treatments, high pressure

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Authors: Mindaugas Liaudanskas, Darius Kviklys, Kristina Zymonė, Raimondas Raudonis, Jonas Viškelis, Norbertas Uselis, Pranas Viškelis, Valdimaras Janulis

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Apples are among the most commonly consumed fruits in the world. Based on data from the year 2014, approximately 84.63 million tons of apples are grown per annum. Apples are widely used in food industry to produce various products and drinks (juice, wine, and cider); they are also used unprocessed. Apples in human diet are an important source of different groups of biological active compounds that can positively contribute to the prevention of various diseases. They are a source of various biologically active substances – especially vitamins, organic acids, micro- and macro-elements, pectins, and phenolic, triterpenic, and other compounds. Triterpenic compounds, which are characterized by versatile biological activity, are the biologically active compounds found in apples that are among the most promising and most significant for human health. A specific analytical procedure including sample preparation and High Performance Liquid Chromatography (HPLC) analysis was developed, optimized, and validated for the detection of triterpenic compounds in the samples of different apples, their peels, and flesh from widespread apple cultivars 'Aldas', 'Auksis', 'Connel Red', 'Ligol', 'Lodel', and 'Rajka' grown in Lithuanian climatic conditions. The conditions for triterpenic compound extraction were optimized: the solvent of the extraction was 100% (v/v) acetone, and the extraction was performed in an ultrasound bath for 10 min. Isocratic elution (the eluents ratio being 88% (solvent A) and 12% (solvent B)) for a rapid separation of triterpenic compounds was performed. The validation of the methodology was performed on the basis of the ICH recommendations. The following characteristics of validation were evaluated: the selectivity of the method (specificity), precision, the detection and quantitation limits of the analytes, and linearity. The obtained parameters values confirm suitability of methodology to perform analysis of triterpenic compounds. Using the optimised and validated HPLC technique, four triterpenic compounds were separated and identified, and their specificity was confirmed. These compounds were corosolic acid, betulinic acid, oleanolic acid, and ursolic acid. Ursolic acid was the dominant compound in all the tested apple samples. The detected amount of betulinic acid was the lowest of all the identified triterpenic compounds. The greatest amounts of triterpenic compounds were detected in whole apple and apple peel samples of the 'Lodel' cultivar, and thus apples and apple extracts of this cultivar are potentially valuable for use in medical practice, for the prevention of various diseases, for adjunct therapy, for the isolation of individual compounds with a specific biological effect, and for the development and production of dietary supplements and functional food enriched in biologically active compounds. Acknowledgements. This work was supported by a grant from the Research Council of Lithuania, project No. MIP-17-8.

Keywords: apples, HPLC, triterpenic compounds, validation

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Authors: Benhouda Afaf, Yahia Massinissa, Paolo Grieco

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Hyoscyamus albus L. is a small genus from Solanaceae family known by its use in old traditional medicine in the east of Algeria. Aim: This study aimed to characterize bioactive molecules from H. albus, evaluate their anticancer activity in several cancer cells and investigate their possible molecular mechanism. Materials and Methods: Different compounds (Peak h of fraction F), (Peak 3 of Fraction F), (Peak 1 of fraction C) were isolated from H.albus L by using high-performance chromatography (HPLC), mass spectrometry (MS) and proton NMR (NMR H1). All isolated compounds were subjected to cytotoxicity and antiproliferative assays against a panel of the four cell lines: DU-145, U-2 OS, U-87 MG and LN-229 cell lines and were determined using MTT assay, Annexin V and propodium iodide were used to evaluate apoptosis. Results: The phytochemical study of H. albus Fractions led to the isolation of quercetin-3-O-β-dglucopyranosyl-( 1 → 6)-β-d-glucopyranosid, N-trans-feruloyltyramine, Hydrocaffeoyl-N8- caffeoylspermidine.The biological results indicated that all cell lines were consistently sensitive to P1 FC in a dose-dependent manner. This difference in cytotoxic sensitivity was more pronounced in osteosarcoma cell line, U-2 OS, when compared to prostate cancer and U-87 MG. Cell viability data also demonstrated that only U-87 MG cells were responsive to treatment with Ph FF. compounds P1 FC and Ph FF have induced necrosis and apoptosis in a large part of LN-229 cells. Conclusion: The overall results of the present study provided evidence that isolated compounds are potential therapeutic entities against cancer.

Keywords: hyoscyamus albus, cancer cells, coumpounds, HPLC

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Authors: S. B. Bhandare, K. S. Laddha

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Podphyllum hexandrum belonging to family berberidaceae has gained attention in phytochemical and pharmacological research as it shows excellent anticancer activity and has been used in treatment of skin diseases, sunburns and radioprotection. Chemically it contains lignans and flavonoids such as kaempferol, quercetin and their glycosides. Objective: To isolate and identify Kaempferol and Quercetin from Podophyllum rhizome. Method: The powdered rhizome of Podophyllum hexandrum was subjected to soxhlet extraction with methanol. This methanolic extract is used to obtain podophyllin. Podohyllin was extracted with ethyl acetate and this extract was then concentrated and subjected to column chromatography to obtain purified kaempferol and quercetin. Result: Isolated kaempferol, quercetin were light yellow and dark yellow in colour respectively. TLC of the isolated compounds was performed using chloroform: methanol (9:1) which showed single band on silica plate at Rf 0.6 and 0.4 for kaempferol and quercetin. UV spectrometric studies showed UV maxima (methanol) at 259, 360 nm and 260, 370 nm which are identical with standard kaempferol and quercetin respectively. Both IR spectra exhibited prominent absorption bands for free phenolic OH at 3277 and 3296.2 cm-1 and for conjugated C=O at 1597 and 1659.7 cm-1 respectively. The mass spectrum of kaempferol and quercetin showed (M+1) peak at m/z 287 and 303.09 respectively. 1H NMR analysis of both isolated compounds exhibited typical four-peak pattern of two doublets at δ 6.86 and δ 8.01 which was assigned to H-3’,5’ and H-2’,6’ respectively. Absence of signals less than δ 6.81 in the 1H NMR spectrum supported the aromatic nature of compound. Kaempferol and Quercetin showed 98.1% and 97% purity by HPLC at UV 370 nm. Conclusion: Easy and simple method for isolation of Kaempferol and Quercetin was developed and their structures were confirmed by UV, IR, NMR and mass studies. Method has shown good reproducibility, yield and purity.

Keywords: flavonoids, kaempferol, podophyllum rhizome, quercetin

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Authors: Gule Teri

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The effective validation of analytical methods forms a crucial component of pharmaceutical manufacturing. However, traditional validation techniques can occasionally fail to fully account for inherent variations within datasets, which may result in inconsistent outcomes. This deficiency in validation accuracy is particularly noticeable when quantifying low concentrations of active pharmaceutical ingredients (APIs), excipients, or impurities, introducing a risk to the reliability of the results and, subsequently, the safety and effectiveness of the pharmaceutical products. In response to this challenge, we introduce an enhanced, tolerance-based Design of Experiments (DoE) approach for the validation of analytical methods. This approach distinctly measures variability with reference to tolerance or design margins, enhancing the precision and trustworthiness of the results. This method provides a systematic, statistically grounded validation technique that improves the truthfulness of results. It offers an essential tool for industry professionals aiming to guarantee the accuracy of their measurements, particularly for low-concentration components. By incorporating this innovative method, pharmaceutical manufacturers can substantially advance their validation processes, subsequently improving the overall quality and safety of their products. This paper delves deeper into the development, application, and advantages of this tolerance-based DoE approach and demonstrates its effectiveness using High-Performance Liquid Chromatography (HPLC) data for verification. This paper also discusses the potential implications and future applications of this method in enhancing pharmaceutical manufacturing practices and outcomes.

Keywords: tolerance-based design, design of experiments, analytical method validation, quality control, biopharmaceutical manufacturing

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Authors: Yonghwa Lee, Yoon Suk Kim, Yongsub Yi

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This study was to confirm the effects of anti-melanogenesis and anti-inflammatory effects from Opuntia humifusa fruit and stem extracts. A potent anti-oxidant activity was shown from the leaf extract at IC50 value of 38.33±1.07 μg/mL and fruit extract at IC50 value of 40.23±2.21 μg/mL by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Also, phenolic contents were confirmed total phenolic assay by high performance liquid chromatography (HPLC). Fraction of taxifolin from leaf extract was identified using HPLC and gas chromatography/mass spectrometry. The extracts of Opuntia humifusa fruit and stem were confirmed about toxicity effect in B16 F1 by cell viability. Melanin contents were decreased. Opuntia humifusa fruit and stem extracts had a positive effect of melanin synthesis inhibition for skin whitening. In investigating the anti-inflammatory activities of Opuntia humifusa, the results of cell viability indicated that taxifolin did not show cytotoxicity on RAW264.7 cells at 500 μM of concentration. The results show that taxifolin inhibited lipopolysaccharide (LPS)-induced production of Nitrite oxide (NO). In addition, taxifolin indicated the inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) -α and interleukin (IL) -6 productions by cytokine assay and cyclooxygenase (COX)-2 expression by western blot analysis, meaning that taxifolin had a significant anti-inflammatory effect. Our results suggested that taxifolin from Opuntia humifusa has anti-melanogenesis and anti-inflammatory activities.

Keywords: anti-melanogenesis, anti-inflammatory, Opuntia humifusa, taxifolin

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Authors: Seema Garcha, Sheetal Chopra, Navraj Sarao

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Plant hormones play a role in internal colonization by beneficial microbes and also systemic acquired resistance. They define qualitative and quantitative nature of root microbiome and also influence dynamics of root rhizospheric soil. The present study is an attempt to relate salicylic acid (signal molecule) content and qualitative nature of root endophytes at various stages in the growth of rice varieties of commercial value- Parmal 121 and Basmati 1121. Root seedlings of these varieties were raised using tissue culture techniques and then they were transplanted in the fields. Cultivation was done using conventional methods in agriculture. Field soil contained 0.39% N, 75.12 Kg/hectare of phosphorus and 163.0 Kg/hectare of potassium. Microfloral profiling of the root tissue was done using the selective microbiological medium. The salicylic acid content was estimated using HPLC-Agilent 1100 HPLC Series. Salicylic acid level of Basmati 1121 remained relatively low at the time of transplant and 90 days after transplant. It increased marginally at 60 days. A similar trend was observed with Parmal 121 as well. However, Parmal variety recorded 0.935 ug/g of salicylic acid at 60 days after transplant. Salicylic acid content decreased after 90 days as both the rice varieties remained disease free. The endophytic root microflora was established by 60 days after transplant in both the varieties after which their population became constant. Rhizobium spp dominated over Azotobacter spp. Genetic profiling of endophytes for nitrogen-fixing ability is underway.

Keywords: plant-microbe interaction, rice, root microbiome, salicylic acid

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Authors: Ali Mashinchian Moradi, Mahmood Sinaei

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Study on the biomarkers to assess health status of marine ecosystems has an important value in biomonitoring of marine environment. Accordingly, accumulation of polycyclic aromatic hydrocarbons in sediment, water and tissues (liver and gill) of mudskipper (Boleophthalmus dussmieri) and some physiological responses like lysosomal membrane change in haemocytes and the Glutathione-S Transferase (GST) activity in the liver were measured in mudskippers. Samples were collected from five sites along the noth western cost of the Persian Gulf. PAHs concentration was measured by HPLC method. The activity of GST enzyme was analysed by spectrophotometric method. Total PAH concentration in coastal seawater, sediments, liver and gill tissues ranged between 0.80-18.34 ug/L, 113.550-3384.34 ng/g dw, 3.99-46.64 ng/g dw and 3.11-17.This study showed that PAH concentrations in this region are not higher than available standards. The findings revile that lysosomal membrane destabilization and liver GST activities are highly sensitive to PAHs in mudskipper, B. dussumieri. Sediment PAH concentrations were strongly correlated with biomarkers, indicating PAHs were biologically available to fish. Thus, mudskipper perceived to be good sentinel organism for PAH pollution biomonitoring.

Keywords: PAHs, biomarker, mudskipper, Persian Gulf

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Authors: Farid Benkaci-Alia, Abdelhamid Neggada, Sophie Laurentb

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A new extraction method of polyphenols from honey using a biodegradable resin was developed and compared with the common commercial resin amberlite XAD2. For this purpose, three honey samples of Algerian origin were selected for the different physico-chemical and biochemical parameters study. After extraction of the target compounds by both resins, the polyphenol content was determined, the antioxidant activity was tested, and LC-MS analyses were performed for identification and quantification. The results showed that physico-chemical and biochemical parameters meet the norms of the International Honey commission, and the H1 sample seemed to be of high quality. The optimal conditions of extraction by biodegradable resin were a pH of 3, an adsorption dose of 40 g/L, a contact time of 50 min, an extraction temperature of 60°C and no stirring. The regeneration and reuse number of both resins was three cycles. The polyphenol contents demonstrated a higher extraction efficiency of biosorbent than of XAD2, especially in H1. LC-MS analyses allowed for the identification and quantification of fifteen compounds in the different honey samples extracted using both resins and the most abundant compound was 3,4,5-trimethoxybenzoic acid. In addition, the biosorbent extracts showed stronger antioxidant activities than the XAD2 extracts.

Keywords: extraction, polyphénols, biosorbent, resin amberlite, HPLC-MS

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Authors: S. Hannah Elizabeth, A. Panneerselvam

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Objective: The main objective was to evaluate the degradative properties of Bacillus cereus sp TUHP2 isolated from TNT-Polluted soils in the Vellore District, Tamil Nadu, India. Methods: Among the 3 bacterial genera isolated from different soil samples, one potent TNT degrading strain Bacillus cereus sp TUHP2 was identified. The morphological, physiological and the biochemical properties of the strain Bacillus cereus sp TUHP2 was confirmed by conventional methods and genotypic characterization was carried out using 16S r-DNA partial gene amplification and sequencing. The broken down by products of DNT in the extract was determined by Gas Chromatogram- Mass spectrometry (GC-MS). Supernatant samples from the broth studied at 24 h interval were analyzed by HPLC analysis and the effect on various nutritional and environmental factors were analysed and optimized for the isolate. Results: Out of three isolates one strain TUHP2 were found to have potent efficiency to degrade TNT and revealed the genus Bacillus. 16S rDNA gene sequence analysis showed highest homology (98%) with Bacillus cereus and was assigned as Bacillus cereus sp TUHP2. Based on the energy of the predicted models, the secondary structure predicted by MFE showed the more stable structure with a minimum energy. Products of TNT Transformation showed colour change in the medium during cultivation. TNT derivates such as 2HADNT and 4HADNT were detected by HPLC chromatogram and 2ADNT, 4ADNT by GC/MS analysis. Conclusion: Hence this study presents the clear evidence for the biodegradation process of TNT by strain Bacillus cereus sp TUHP2.

Keywords: bioremediation, biodegradation, biotransformation, sequencing

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Authors: Faisal A. Salih

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Verapamil hydrochloride (Ver) is a drug used in medicine for arrythmia, angina and hypertension as a calcium channel blocker. For the quantitative determination of Ver in dosage forms, the HPLC method is most often used. A convenient alternative to the chromatographic method is potentiometry using a Verselective electrode, which does not require expensive equipment, can be used without separation from the matrix components, which significantly reduces the analysis time, and does not use toxic organic solvents, being a "green", "environmentally friendly" technique. It has been established in this study that the rational choice of the membrane plasticizer and the preconditioning and measurement algorithms, which prevent nonexchangeable extraction of Ver into the membrane phase, makes it possible to achieve excellent analytical characteristics of Ver-selective electrodes based on commercially available components. In particular, an electrode with the following membrane composition: PVC (32.8 wt %), ortho-nitrophenyloctyl ether (66.6 wt %), and tetrakis-4-chlorophenylborate (0.6 wt % or 0.01 M) have the lower detection limit 4 × 10−8 M and potential reproducibility 0.15–0.22 mV. Both direct potentiometry (DP) and potentiometric titration (PT) methods can be used for the determination of Ver in tablets and injection solutions. Masses of Ver per average tablet weight determined by the methods of DP and PT for the same set of 10 tablets were (80.4±0.2 and80.7±0.2) mg, respectively. The masses of Ver in solutions for injection, determined by DP for two ampoules from one set, were (5.00±0.015 and 5.004±0.006) mg. In all cases, good reproducibility and excellent correspondence with the declared quantities were observed.

Keywords: verapamil, potentiometry, ion-selective electrode, pharmaceutical analysis

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Authors: Ahlem Nefzi, Rania Aydi Ben Abdallah, Hayfa Jabnoun-Khiareddine, Nawaim Ammar, Sined Medimagh-Saidana, Mejda Daami-Remadi

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In the present study, leaf, stem, and fruit aqueous extracts of native wild Solanum linnaeanum L. were screened for their ability to suppress Fusarium Crown and Root Rot disease and to enhance tomato (Solanum lycopersicum L.) growth under greenhouse conditions. Leaf extract used at 30% w/v was the most effective in reducing leaf and root damage index by 92.3% and the extent of vascular discoloration by 97.56% compared to Fusarium oxyxporum f. sp radicis lycopersici -inoculated and untreated control. A significant promotion of growth parameters (root length, shoot height, root and shoot biomass and stem diameter) was recorded on tomato cv. Rio Grande seedlings by 40.3-94.1% as compared to FORL inoculated control and by 9.6-88.8% over pathogen-free control. All S. linnaeanum aqueous extracts tested significantly stimulated the germination by 10.2 to 80.1% relative to the untreated control. FORL mycelial growth, assessed using the poisoned food technique, varied depending on plant organs, extracts, and concentrations used. Butanolic extracts were the most active, leading to 60.81% decrease in FORL mycelial growth. HPLC analysis of butanolic extract revealed the presence of thirteen phenolic compounds. Thus, S. linnaeanum can be explored as a potential natural source of antifungal and biofertilizing compounds.

Keywords: antifungal activity, HPLC-MS analysis, Fusarium oxysporum f. sp. radicis-lycopersici, tomato growth

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Authors: Nji Queenta Ngum, Mwanza Mulunda

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Introduction Most African research reports on the frequent aflatoxin contamination of various foodstuffs, with researchers rarely specifying which of the Aspergillus species are present in these commodities. Numerous research works provide evidence of the ability of fungi to grow, thrive, and interact with other crop species and focus on the fact that these processes are largely affected by climatic variables. South Africa is a water-stressed country with high spatio-temporal rainfall variability; moreover, temperatures have been projected to rise at a rate twice the global rate. This weather pattern change may lead to crop stress encouraging mold contamination with subsequent mycotoxin production. In this study, the biodiversity and distribution of Aspergillus species with their corresponding toxins in maize from six distinct maize producing regions with different weather patterns in South Africa were investigated. Materials And Methods By applying cultural and molecular methods, a total of 1028 maize samples from six distinct agro-climatic regions were examined for contamination by the Aspergillus species while the high performance liquid chromatography (HPLC) method was applied to analyse the level of contamination by aflatoxins. Results About 30% of the overall maize samples were contaminated by at least one Aspergillus species. Less than 30% (28.95%) of the 228 isolates subjected to the aflatoxigenic test was found to possess at least one of the aflatoxin biosynthetic genes. Furthermore, almost 20% were found to be contaminated with aflatoxins, with mean total aflatoxin concentration levels of 64.17 ppb. Amongst the contaminated samples, 59.02% had mean total aflatoxin concentration levels above the SA regulatory limit of 20ppb for animals and 10 for human consumption. Conclusion In this study, climate variables (rainfall reduction) were found to significantly (p<0.001) influence the occurrence of the Aspergillus species (especially Aspergillus fumigatus) and the production of aflatoxin in South Africa commercial maize by maize variety, year of cultivation as well as the agro-climatic region in which the maize is cultivated. This included, amongst others, a reduction in the average annual rainfall of the preceding year to about 21.27 mm, and, as opposed to other regions whose average maximum rainfall ranged between 37.24 – 44.1 mm, resulted in a significant increase in the aflatoxin contamination of maize.

Keywords: aspergillus species, aflatoxins, diversity, drought, food safety, HPLC and PCR techniques

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Authors: Attila Kiss, Erzsébet Némedi, Zoltán Naár

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As natural prebiotic (non-digestible) carbohydrates stimulate the growth of colon microflora and contribute to maintain the health of the host, analytical studies aiming at revealing the chemical behavior of these beneficial food components came to the forefront of interest. Food processing (especially baking) may lead to a significant conversion of the parent compounds, hence it is of utmost importance to characterize the transformation patterns and the plausible decomposition products formed by thermal degradation. The relevance of this work is confirmed by the wide-spread use of these carbohydrates (fructo-oligosaccharides, cyclodextrins, raffinose and resistant starch) in the food industry. More and more functional foodstuffs are being developed based on prebiotics as bioactive components. 12 different types of oligosaccharides have been investigated in order to reveal their thermal degradation characteristics. Different carbohydrate derivatives (D-fructose and D-glucose oligomers and polymers) have been exposed to elevated temperatures (150 °C 170 °C, 190 °C, 210 °C, and 220 °C) for 10 min. An advanced HPLC method was developed and used to identify the decomposition products of carbohydrates formed as a consequence of thermal treatment. Gradient elution was applied with binary solvent elution (acetonitrile, water) through amine based carbohydrate column. Evaporative light scattering (ELS) proved to be suitable for the reliable detection of the UV/VIS inactive carbohydrate degradation products. These experimental conditions and applied advanced techniques made it possible to survey all the formed intermediers. Change in oligomer distribution was established in cases of all studied prebiotics throughout the thermal treatments. The obtained results indicate increased extent of chain degradation of the carbohydrate moiety at elevated temperatures. Prevalence of oligomers with shorter chain length and even the formation of monomer sugars (D-glucose and D-fructose) might be observed at higher temperatures. Unique oligomer distributions, which have not been described previously are revealed in the case of each studied, specific carbohydrate, which might result in various prebiotic activities. Resistant starches exhibited high stability when being thermal treated. The degradation process has been modeled by a plausible reaction mechanism, in which proton catalyzed degradation and chain cleavage take place.

Keywords: prebiotics, thermal degradation, fructo-oligosaccharide, HPLC, ELS detection

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Authors: Leila Najafian

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Sarcoplasmic protein hydrolysates (SPHs) and myofibrillar protein hydrolysates (MPHs) from patin (Pangasius sutchi) were produced using two types of proteases: Papain and Alcalase. 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging activities and metal chelating activity assays for antioxidant activities were carried out on the SPHs and MPHs. The hydrolysates were isolated and purified by ultrafiltration, gel filtration and reverse phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) was used in identifying peptide sequences. The results showed that when the DH of MPHs increased, the protein solubility increased, while the highest amount of the protein solubility of SPHs was after 60 min incubation. The effect of DH on antioxidant activities of SPHs and MPHs was investigated. Among the hydrolysates, papain-MPH and Alcalase-SPH, which had the highest antioxidant activities, were purified. The potent fractions obtained from RP-HPLC of sarcoplasmic (SI 3 fraction) and myofibrillar (MI 4 fraction) hydrolysates showed the highest DPPH radical scavenging activity. The FVNQPYLLYSVHMK peptide for MPH and the LVVDIPAALQHA peptide for SPH exhibited the highest antioxidant activity. The presence of hydrophobic and hydrophilic amino acids, namely leucine (L), valine (V), phenylalanine (F), histidine (H) and proline (P), in the peptide sequences of SPH and MPH are believed to contribute to high antioxidant activity. Hence, SPH and MPH from patin have the potential as a natural functional ingredient in food and pharmaceutical industry.

Keywords: patin (Pangasius sutchi), protein hydrolysates, antioxidative peptides, mass spectrometry

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Authors: Nadeem A. Siddique, Mohd Mujeeb, Kahkashan

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Introduction: The objective of the projected work is to study the occurrence of the aflatoxins B1, B2, G1and G2 in remedial plants, exclusively in Delphinium denudatum. The aflatoxins were analysed by high-performance liquid chromatography–tandem quadrupole mass spectrometry with electrospray ionization (HPLC–MS/MS) and immunoaffinity column chromatography were used for extraction and purification of aflatoxins. PDA media was selected for fungal count. Results: A good quality linear relationship was originated for AFB1, AFB2, AFG1 and AFG2 at 1–10 ppb (r > 0.9995). The analyte precision at three different spiking levels was 88.7–109.1 %, by means of low per cent relative standard deviations in each case. Within 5 to7 min aflatoxins can be separated using an Agilent XDB C18-column. We found that AFB1 and AFB2 were not found in D. denudatum. This was reliable through exceptionally low figures of fungal colonies observed after 6 hr of incubation. The developed analytical method is straightforward, be successfully used to determine the aflatoxins. Conclusion: The developed analytical method is straightforward, simple, accurate, economical and can be successfully used to find out the aflatoxins in remedial plants and consequently to have power over the quality of products. The presence of aflatoxin in the plant extracts was interrelated to the least fungal load in the remedial plants examined.

Keywords: aflatoxins, delphinium denudatum, liquid chromatography, mass spectrometry

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Authors: Kyung-Soon Choi, Ji-Young Hwang, Young-Hee Pyo

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Vinegars have been used as an alternative remedy for treating gout, but the scientific basis remains to be elucidated. In this study, acetic acid fermentation was applied for the first time to Monascus-fermented soybeans to examine its effect on the bioactive components together with the xanthine oxidase inhibitory (XOI) activity of the soy vinegar. The content of total phenols (0.47~0.97 mg gallic acid equivalents/mL) and flavonoids (0.18~0.39 mg quercetin equivallents/mL) were spectrophotometrically determined, and the content of organic acid (10.22~59.76 mg/mL) and isoflavones (6.79~7.46 mg/mL) were determined using HPLC-UV. The analytical method for ubiquinones (0.079~0.276 μg/mL) employed saponification before solvent extraction and quantification using LC-MS. Soy vinegar also showed significant XOI (95.3%) after 20 days of acetic acid fermentation at 30 °C. The results suggest that soy vinegar has potential as a novel medicinal food.

Keywords: acetic acid fermentation, bioactive component, soy vinegar, xanthine oxidase inhibitory activity

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Authors: F. Portet-Koltalo, Y. Tian, I. Berger, C. Boulanger-Lecomte, A. Benamar, N. Machour

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Sediments are complex environments which can accumulate a great variety of persistent toxic contaminants such as polychlorobiphenyles (PCBs), polycyclic aromatic hydrocarbons (PAHs) and some of their more toxic degradation metabolites such as hydroxylated PAHs (OH-PAHs). Owing to their composition, fine clayey sediments can be more difficult to extract than soils using conventional solvent extraction processes. So this study aimed to compare the potential of MSPD (matrix solid phase dispersive extraction) to extract PCBs, PAHs and OH-PAHs, in comparison with microwave assisted extraction (MAE). Methodologies: MAE extraction with various solvent mixtures was used to extract PCBs, PAHs and OH-PAHs from sediments in two runs, followed by two GC-MS analyses. MSPD consisted in crushing the dried sediment with dispersive agents, introducing the mixture in cartridges and eluting the target compounds with an appropriate volume of selected solvents. So MSPD combined with cartridges containing MIPs (molecularly imprinted polymers) designed for OH-PAHs was used to extract the three families of target compounds in only one run, followed by parallel analyses in GC-MS for PAHs/PCBs and HPLC-FLD for OH-PAHs. Results: MAE extraction was optimized to extract from clayey sediments, in two runs, PAHs/PCBs in one hand and OH-PAHs in the other hand. Indeed, the best conditions of extractions (mixtures of extracting solvents, temperature) were different if we consider the polarity and the thermodegradability of the different families of target contaminants: PAHs/PCBs were better extracted using an acetone/toluene 50/50 mixture at 130°C whereas OH-PAHs were better extracted using an acetonitrile/toluene 90/10 mixture at 100°C. Moreover, the two consecutive GC-MS analyses contributed to double the total analysis time. A matrix solid phase dispersive (MSPD) extraction procedure was also optimized, with the first objective of increasing the extraction recovery yields of PAHs and PCBs from fine-grained sediment. The crushing time (2-10 min), the nature of the dispersing agents added for purifying and increasing the extraction yields (Florisil, octadecylsilane, 3-chloropropyle, 4-benzylchloride), the nature and the volume of eluting solvents (methylene chloride, hexane, hexane/acetone…) were studied. It appeared that in the best conditions, MSPD was a better extraction method than MAE for PAHs and PCBs, with respectively, mean increases of 8.2% and 71%. This method was also faster, easier and less expensive. But the other advantage of MSPD was that it allowed to introduce easily, just after the first elution process of PAHs/PCBs, a step permitting the selective recovery of OH-PAHs. A cartridge containing MIPs designed for phenols was coupled to the cartridge containing the dispersed sediment, and various eluting solvents, different from those used for PAHs and PCBs, were tested to selectively concentrate and extract OH-PAHs. Thereafter OH-PAHs could be analyzed at the same time than PAHs and PCBs: the OH-PAH extract could be analyzed with HPLC-FLD, whereas the PAHs/PCBs extract was analyzed with GC-MS, adding only few minutes more to the total duration of the analytical process. Conclusion: MSPD associated to MIPs appeared to be an easy, fast and low expensive method, able to extract in one run a complex mixture of toxic apolar and more polar contaminants present in clayey fine-grained sediments, an environmental matrix which is generally difficult to analyze.

Keywords: contaminated fine-grained sediments, matrix solid phase dispersive extraction, microwave assisted extraction, molecularly imprinted polymers, multi-pollutant analysis

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Authors: N. Yang, R. Linforth, I. Fisk

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The influence of food processing and choice of flavour solvent was investigated using biscuits prepared with vanillin flavour as an example. Powder vanillin either was added directly into the dough or dissolved into flavour solvent then mixed into the dough. The impact of two commonly used flavour solvents on food quality was compared: propylene glycol (PG) or triacetin (TA). The analytical approach for vanillin detection was developed by chromatography (HPLC-PDA), and the standard extraction method for vanillin was also established. The results indicated the impact of solvent choice on vanillin level during biscuit preparation. After baking, TA as a more heat resistant solvent retained more vanillin than PG, so TA is a better solvent for products that undergo a heating process. The results also illustrated the impact of mixing and baking on vanillin stability in the matrices. The average loss of vanillin was 33% during mixing and 13% during baking, which indicated that the binding of vanillin to fat or flour before baking might cause larger loss than evaporation loss during baking.

Keywords: biscuit, flavour stability, food quality, vanillin

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Authors: María José Patiño-Ropero, Manuel Alcamí, Al Mokhtar Lamsabhi, José Luis Alonso-Prados, Pilar Sandín-España

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Tralkoxydim, commercialized under different trade names, among them Splendor® (25% active ingredient), is a cyclohexanedione herbicide used in wheat and barley fields for the post-emergence control of annual winter grass weeds. Due to their physicochemical properties, herbicides belonging to this family are known to be susceptible to reaching natural waters, where different degradation pathways can take place. Photolysis represents one of the main routes of abiotic degradation of these herbicides in water. This transformation pathway can lead to the formation of unknown by-products, which could be more toxic and/or persistent than the active substances themselves. Therefore, there is a growing need to understand the science behind such dissipation routes, which is key to estimating the persistence of these compounds and ensuring the accurate assessment of environmental behavior. However, to our best knowledge, any information regarding the photochemical behavior of tralkoxydim under natural conditions in an aqueous environment has not been available till now in the literature. This work has focused on investigating the photochemical behavior of tralkoxydim herbicide and its commercial formulation (Splendor®) in the ultrapure, river and spring water using simulated solar radiation. Besides, the evolution of detected degradation products formed in the samples has been studied. A reversed-phase HPLC-DAD (high-performance liquid chromatography with diode array detector) method was developed to evaluate the kinetic evolution and to obtain the half-lives. In both cases, the degradation rates of active ingredient tralkoxydim in natural waters were lower than in ultrapure water following the order; river water < spring water < ultrapure water, and with first-order half-life values of 5.1 h, 2.7 h and 1.1 h, respectively. These findings indicate that the photolytical behavior of active ingredients is largely affected by the water composition, and these components can exert an internal filter effect. In addition, tralkoxydim herbicide and its formulation showed the same half-lives for each one of the types of water studied, showing that the presence of adjuvants in the commercial formulation has not any effect on the degradation rates of the active ingredient. HPLC-MS (high-performance liquid chromatography with mass spectrometry) experiments were performed to study the by-products deriving from the photodegradation of tralkoxydim in water. Accordingly, three compounds were tentatively identified. These results provide a better understanding of the tralkoxydim herbicide behavior in natural waters and its fate in the environment.

Keywords: by-products, natural waters, photodegradation, tralkoxydim herbicide

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Authors: Ajay Kumar, Satwinderjeet Kaur

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Onosma bracteata Wall. (Boraginaceae), is known to be a medicinal plant, useful in the treatment of body swellings, abdominal pain and urinary calculi, etc. The present study focused on the radical scavenging and cancer growth inhibitory properties of isolates from O. bracteata. Obea fraction demonstrated noticeable free radical scavenging ability along with antiproliferative activity in human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung cancer A549 cell lines using MTT assay with GI50 values of 88.56, 101.61 and 112.7 μg/ml, respectively. The scanning electron and confocal microscopy studies showed morphological alterations including nuclear condensation and formation of apoptotic bodies in osteosarcoma MG-63 cells. Obea fraction in osteosarcoma MG-63 cells augmented the reactive oxygen species (ROS) level and decreased the mitochondrial membrane potential. Flow cytometry analysis revealed the Obea treated cells to be arrested in the G0/G1 phase in a dose dependent manner supported by the observed increase in the early apoptotic cell population. Western blotting analysis showed that the expression of p-NF-kB, COX-2, p-Akt, and Bcl-xL decreased whereas, the expression of GSK-3β, p53, caspase-3 and caspase-9 proteins increased. The downregulation of Bcl-2, Cyclin E, CDK2 and mortalin gene expression and upregulation of p53 genes was unfolded in RT-qPCR studies. The presence of catechin, kaempferol, Onosmin A and epicatechin, as revealed in high-performance liquid chromatography (HPLC) studies, contributes towards the chemopreventive potential of O. bracteata which can be tapped for chemotherapeutic use.

Keywords: apoptosis, confocal microscopy, HPLC, mitochondria membrane potential, reactive oxygen species

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Authors: Jasna Čanadanović-Brunet, Gordana Ćetković, Sonja Djilas, Vesna Tumbas-Šaponjac, Jelena Vulić, Sladjana Stajčić

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Honey, produced by the honeybee, is a natural saturated sugar solution, which is mainly composed of a complex mixture of carbohydrates. Besides this, it also contains certain minor constituents, proteins, enzymes, amino and organic acids, lipids, vitamins, phenolic acids, flavonoids and carotenoids. Honey serves as a source of natural antioxidants, which are effective in reducing the risk of heart disease, cancer, immune-system decline, cataracts, and different inflammatory processes. Honey is consumed in its natural form alone, but also in combination with nuts and various kinds of dried fruits (plums, figs, cranberries, apricots etc.). The aim of this research was to investigate the contribution of dried apricot addition to polyphenols and flavonoids contents and antioxidant activities of honey. Some individual phenolic compounds in Serbian polyfloral honey (PH), linden honey (LH) and also in their mixtures with dried apricot, in 40% mass concentrations (PH40; LH40), were identified and quantified by HPLC. The most dominant phenolic compound was: gallic acid in LH (11.14 mg/100g), LH40 (42.65 mg/100g), PH (7.24 mg/100g) and catehin in PH40 (11.83 mg/100g). The antioxidant activity of PH, LH, PH40 and LH40 was tested by measuring their ability to scavenge hydroxyl radicals (OH) by electron spin resonance spectroscopy (ESR). Honey samples with 40% dried apricot exhibited better antioxidant activity measured by hydroxyl radical scavenging activity. The EC50 values, the amount of antioxidant necessary to decrease the initial concentration of OH radicals by 50%, were: EC50PH=3.36 mg/ml, EC50LH=13.36 mg/ml, EC50PH40=2.29 mg/ml, EC50 LH40=7.78 mg/ml. Our results indicate that supplementation of polyfloral honey and linden honey with dried apricots improves antioxidant activity of honey by enriching the phenolic composition.

Keywords: honey, dried apricot, HPLC, hydroxyl radical

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Authors: Yasinta Ratna Esti Wulandari, Vivitri Dewi Prasasty, Adrianus Rio, Cindy Geniola

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Mulberry is a plant that widely cultivated around the world, mostly for silk industry. In recent years, the study showed that the mulberry leaves have an anti-diabetic effect which mostly comes from the compound known as 1-deoxynojirimycin (DNJ). DNJ is a very potent α-glucosidase inhibitor. It will decrease the degradation rate of carbohydrates in digestive tract, leading to slower glucose absorption and reducing the post-prandial glucose level significantly. The mulberry leaves also known as the best source of DNJ. Since then, the DNJ in mulberry leaves had received a considerable attention, because of the increased number of diabetic patients and the raise of people awareness to find a more natural cure for diabetic. The DNJ content in mulberry leaves varied depend on the mulberry species, leaf’s age, and the plant’s growth environment. Few of the mulberry varieties that were cultivated in Indonesiaare Morus alba var. kanva-2, M. alba var. multicaulis, M. bombycis var. lembang, and M. cathayana. The lack of data concerning phytochemicals contained in the Indonesian mulberry leaves are restraining their use in the medicinal field. The aim of this study is to fully utilize the use of mulberry leaves cultivated in Indonesia as a medicinal herb in local, national, or global community, by determining the DNJ and other phytochemical contents in them. This study used eight leaf samples which are the young leaves and mature leaves of both Morus alba var. kanva-2, M. alba var. multicaulis, M. bombycis var. lembang, and M. cathayana. The DNJ content was analyzed using reverse phase high performance liquid chromatography (HPLC). The stationary phase was silica C18 column and the mobile phase was acetonitrile:acetic acid 0.1% 1:1 with elution rate 1 mL/min. Prior to HPLC analysis the samples were derivatized with FMOC to ensure the DNJ detectable by VWD detector at 254 nm. Results showed that the DNJ content in samples are ranging from 2.90-0.07 mg DNJ/ g leaves, with the highest content found in M. cathayana mature leaves (2.90 ± 0.57 mg DNJ/g leaves). All of the mature leaf samples also found to contain higher amount of DNJ from their respective young leaf samples. The phytochemicals in leaf samples was tested using qualitative test. Result showed that all of the eight leaf samples contain alkaloids, phenolics, flavonoids, tannins, and terpenes. The presence of this phytochemicals contribute to the therapeutic effect of mulberry leaves. The pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) analysis was also performed to the eight samples to quantitatively determine their phytochemicals content. The pyrolysis temperature was set at 400 °C, with capillary column Phase Rtx-5MS 60 × 0.25 mm ID stationary phase and helium gas mobile phase. Few of the terpenes found are known to have anticancer and antimicrobial properties. From all the results, all of four samples of mulberry leaves which are cultivated in Indonesia contain DNJ and various phytochemicals like alkaloids, phenolics, flavonoids, tannins, and terpenes which are beneficial to our health.

Keywords: Morus, 1-deoxynojirimycin, HPLC, Py-GC-MS

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Authors: Dandan Shen, Hui-zhu Wang, Xi Yu, LiLi Gui, Xiao Wei, Xiao-yan Jiang, Da-wei Wang, Min Hong

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Yu-ping-feng-san (YPFS) is a clinical medicine for asthma and other allergic diseases, but the mechanism of YPFS on relapse of allergy is unclear. Currently, people come to realize the epithelial cells(EC) play a key role in stimulating and regulating local immune response. The study of thymic stromal lymphopoietin（TSLP derived from EC provides an important evidence that the EC can regulate immune response to stimulate allergic response. In this study, we observed the effect of YPFS on TSLP in vivo and in vitro. We established a method by using bronchial epithelial cells (16HBE) for screening potential bioactive components by HPLC-MS in YPFS and then analyzed the components in serum containing YPFS by UPLC-MS. The results showed that YPFS could decrease TSLP protein level in OVA-sensitized mice and 16HBE cells. Five components combing with the 16HBE cells were both detected in the serum.

Keywords: TSLP, bronchial epithelial cells, cell-binding, drug-containing serum

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Authors: Farideh Tabatabaee Yazdi, Fereshteh Falah, Alireza Vasiee

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Introduction: Gamma-aminobutyric acid (GABA) is a non-protein amino acid that is widely present in organisms. GABA is a kind of pharmacological and biological component and its application is wide and useful. Several important physiological functions of GABA have been characterized, such as neurotransmission and induction of hypotension. GABA is also a strong secretagogue of insulin from the pancreas and effectively inhibits small airway-derived lung adenocarcinoma and tranquilizer. Many microorganisms can produce GABA, and lactic acid bacteria have been a focus of research in recent years because lactic acid bacteria possess special physiological activities and are generally regarded as safe. Among them, the Lb. Brevis produced the highest amount of GABA. The major factors affecting GABA production have been characterized, including carbon sources and glutamate concentration. The use of food industry waste to produce valuable products such as amino acids seems to be a good way to reduce production costs and prevent the waste of food resources. In a dairy factory, a high volume of sludge is produced from a separator that contains useful compounds such as growth factors, carbon, nitrogen, and organic matter that can be used by different microorganisms such as Lb.brevis as carbon and nitrogen sources. Therefore, it is a good source of GABA production. GABA is primarily formed by the irreversible α-decarboxylation reaction of L-glutamic acid or its salts, catalysed by the GAD enzyme. In the present study, this aim was achieved for the fast-growing of Lb.brevis and producing GABA, using the dairy industry sludge as a suitable growth medium. Lactobacillus Brevis strains obtained from Microbial Type Culture Collection (MTCC) were used as model strains. In order to prepare dairy sludge as a medium, sterilization should be done at 121 ° C for 15 minutes. Lb. Brevis was inoculated to the sludge media at pH=6 and incubated for 120 hours at 30 ° C. After fermentation, the supernatant solution is centrifuged and then, the GABA produced was analyzed by the Thin Layer chromatography (TLC) method qualitatively and by the high-performance liquid chromatography (HPLC) method quantitatively. By increasing the percentage of dairy sludge in the culture medium, the amount of GABA increased. Also, evaluated the growth of bacteria in this medium showed the positive effect of dairy sludge on the growth of Lb.brevis, which resulted in the production of more GABA. GABA-producing LAB offers the opportunity of developing naturally fermented health-oriented products. Although some GABA-producing LAB has been isolated to find strains suitable for different fermentations, further screening of various GABA-producing strains from LAB, especially high-yielding strains, is necessary. The production of lactic acid, bacterial gamma-aminobutyric acid, is safe and eco-friendly. The use of dairy industry waste causes enhanced environmental safety. Also provides the possibility of producing valuable compounds such as GABA. In general, dairy sludge is a suitable medium for the growth of Lactic Acid Bacteria and produce this amino acid that can reduce the final cost of it by providing carbon and nitrogen source.

Keywords: GABA, Lactobacillus, HPLC, dairy sludge

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