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   Table of Contents - Current issue
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January-March 2021
Volume 20 | Issue 1
Page Nos. 1-103

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ORIGINAL ARTICLE  

Rapid quantitative estimation of metformin and ertugliflozin in rat plasma by liquid chromatography-tandam mass spectroscopy and its application to pharmacokinetic studies Highly accessed article p. 1
P Venkateswarao Rao, A Lakshmana Rao, SVUM Prasad
DOI:10.4103/epj.epj_14_20  
Background The development of sound bioanalytical liquid chromatography-mass spectroscopy (LC-MS) method(s) is of paramount importance during the process of drug discovery and development, eventually culminating in marketing approval. The use of oral antidiabetic agents has been increased significantly from past decades, and till now, no bioanalytical method is available for quantitation of metformin (MET) and ertugliflozin (ERT) in the biological matrix that can be applied in bioequivalence studies using LC-MS/MS. Objective To study the use of highly responsive simple liquid–liquid extraction method development using deuterated MET and deuterated ERT, LC-MS/MS method for gradation of MET and ERT in the rat plasma. Materials and methods The chromatographic condition involves isocratic mode using Waters XBridge C18 3.5 μ (150×4.6 mm) column. Mobile phase was 0.1% orthophosphoric acid and acetonitrile in the ratio of 80 : 20 v/v. Detection was carried out on a triple quadrapole MS employing electrospray ionization technique, operating multiple reactions, monitoring with the transitions of m/z 258.2→174.1, m/z 250.1→210.2, m/z 258.2→174.1, and m/z 260.3→210.2 for MET, ERT, deuterated MET, and deuterated ERT, respectively, in the positive ion mode. Results and conclusion The method has been validated, and the linearity was observed in the range of 10–150 ng/ml and 0.1–1.5 ng/ml for MET and ERT, respectively. For intraday and interday %RSD, the values were found to be within the acceptable limits. Recovery studies for MET and ERT obtained, mean recovery of 99.5 and 98.6%, respectively. A battery of stability studies like bench-top stability, autosampler stability, freeze-thaw stability, and long-term stability were performed. Highly responsive simple LC-tandem MS assay method was developed and witnessed for the gradation of MET and ERT in the rat plasma; the developed method was applied to pharmacokinetic studies.
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ORIGINAL ARTICLE Top

Utilization of orange pulp and corn steep liquor for L-methioninase production by Wickerhamomyces subpelliculosus p. 8
Amany A Hassabo, Elsayed E Mostafa, Moataza M Saad, Mohsen H Selim
DOI:10.4103/epj.epj_2_20  
Background and objective L-methioninase has attracted much attention with respect to its proposed applications in both pharmaceuticals and food industry. The aim of this study was to develop an economic medium formulation using agro-industrial by-products as substrates for large-scale production of L-methioninase. Materials and methods Identification of a high L-methioninase-producing yeast isolate was carried out using 18S rRNA molecular technique. Screening of various agro-industrial by-products and optimization of different process parameters were investigated. Partial purification and characterization of a crude enzyme were carried out. Results and discussion A high L-methioninase-producing yeast isolate was phylogenetically identified as Wickerhamomyces subpelliculosus. Among different agro-industrial by-products tested, orange pulp supported maximum enzyme production (94.08 U/ml) followed by cane and beet molasses. In addition, corn steep liquor (CSL) gave high enzyme level (141.12 U/ml) and could be used as an inexpensive alternate for yeast extract. The optimum growth conditions were found to be orange pulp 30% (w/v), CSL 4% (v/v), CaCl2 0.05%, and KH2PO4 0.05% (w/v) at pH 6.0 after 48 h of incubation. This developed medium formulation increased L-methioninase production (161.95 U/ml) by twofold compared with that obtained by the Czapek–Dox’s medium (73.92 U/ml). Crude enzyme was partially purified by heat treatment at 70°C with 2.9 purification fold. The enzyme activity was optimal at temperature 60°C and pH 7.0. The results showed that a mixed formulation of orange pulp and CSL can be used as an effective and economic substrate for the production of L-methioninase by W. subpelliculosus.
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Effect of licorice root and cabbage leaf extracts as a natural fertilizer on growth and productivity of Cynara cardunculus L p. 17
Ahmed E El-Gohary, Hend El-Sayed Wahba, Saber Fayez Hendawy, Mohamed Salah Hussein
DOI:10.4103/epj.epj_13_20  
Background There is an ongoing need to find safe natural sources of plant nutrients. Licorice root and cabbage leaf extracts are being used as sources that can be used for growth and yield of crops to substitute inorganic fertilizers. Objective To study the effect of extracts of cabbage leaves (waste) and licorice roots on Cynara cardunculus L. Materials and methods This experiment was carried out during two seasons (2017/2018 and 2018/2019) at Aladlya Field, Sekem, Sharkia Governorate, Egypt, to study the influence of some plant extracts, that is cabbage leaves’ extract at 0, 1, 2, and 3 g/l as well as licorice root’s extract at 0, 5, 10, and 15 g/l, on growth, yield, and chemical constituents (NPK, total phenolic content, and phenolic compounds) of C. cardunculus L. plants. Results and conclusion Both licorice root and cabbage leaf extracts had positive effects compared with control. However, licorice root extract had more effect on C. cardunculus L. plants compared with cabbage leaves’ extract. The main phenolic compounds were apigeni-7-glucoside (50.9272–161.8283 μg/g), rutin (79.8306–152.3828 μg/g), chlorogenic (4.5107–25.7202 μg/g).
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Statistical optimization of lipase production in solid-state fermentation by Aspergillus tamarii NDA03a and application of the fermented solid as a biocatalyst for biodiesel production p. 23
Hanan M Ahmed, Sayeda S Mohamed, Maysa E Moharam, Magda A El-bendary, Hisham A Abd El-lateaf, Hala A Amin
DOI:10.4103/epj.epj_30_20  
Background and objective Biodiesel, an attractive alternative fuel, is defined by the American Society for Testing and Materials (ASTM) as fatty acid methyl esters (FAME). Biodiesel is an ecofriendly fuel compared with many other transportation fuels. The aim of this study was to implement the statistical approaches for optimization of Aspergillus tamarii NDA03a mutant G lipase produced in solid-state fermentation (SSF), and then application of the dried fermented solid as a biocatalyst for biodiesel production from waste frying oil (WFO). Materials and methods A. tamarii NDA03a mutant (3G) was previously selected as a good lipase producer. Five oil residue meals were evaluated in the presence of wheat bran (WB) for their potential as enzyme inducers and substrates for the production of 3G lipase by SSF. The best oil residue meal was selected and used in subsequent experiments. The fermented solid thus obtained was collected, lyophilized, and used as a biocatalyst for waste frying oil transesterification to FAME. To optimize SSF conditions for lipase production using 3G, a Plackett–Burman design was used at first to screen the critical factors from several process variables, and finally, a central composite design was applied to further estimate the relationship between the variables and response as well as optimize the levels. Response was measured in terms of FAME yield. To verify the adequacy and accuracy of the model, validation experiments were also carried out. Results and conclusion The most favorable oil residue meal that enhances 3G lipase production by SSF was black cumin meal. Results of the Plackett–Burman design revealed that the factors contributing to the main effect were incubation temperature, incubation period, and moisture content. The optimal SSF conditions for lipase production were WB 10 g, black cumin meal 6% (w/w of WB), pH 8, temperature 28°C, moisture content 40%, molasses 1% (w/w of WB), and incubation period 3 days. Under these optimized conditions, produced FAME yield (65.55%) increased by 58% compared with the basal medium (41.46%). A good agreement between the experimental (65.55%) and predicted (65.03%) values was detected. The significance of this model was confirmed by its probability value and lack of fit (P<0.05) and clearly showed that the model was sufficient to describe the correlation between the FAME yield and the tested variables. The obtained results ascertained the success of response surface methodology as an efficient technique to optimize the lipase production in SSF and consequently the ability of application of the dried fermented solid as a biocatalyst for biodiesel production.
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Chemical and biological studies on Moringa oleifera L. cultivated in Egypt p. 33
Mona A Mohamed, Magda T Ibrahim, Nahla S Abdel-Azim, Mostafa M El-Missiry
DOI:10.4103/epj.epj_37_20  
Background Nowadays, Moringa oleifera is being cultivated in Egypt on a wide scale owing to its proven medicinal and economic benefits. Objective The aim was to estimate quantitatively different phenolic acids and flavonoids in the M. oleifera L. leaves. Moreover, gas chromatography–mass spectrometry (GC/MS) analysis was carried out to evaluate seed oil obtained by three different methods of extraction. Different plant extracts were tested for their hepatoprotective, anticancer, and antibacterial activities. Materials and methods Quantification of different phenolic acids and flavonoids in M. oleifera L. leaves was carried out using HPLC. GC/MS was used to determine fatty acid methyl esters of M. oleifera L. seed oil, extracted by three different methods (cold press, solvent extraction, and ultrasound-assisted extraction). Moreover, in-vitro investigations of hepatoprotective, anticancer, and antibacterial activities were done. Results and conclusion HPLC profiling of leaves extract indicated that ellagic acid is the major phenolic acid (120.15 mg/g). Quercetin and rutin were recorded as major flavonoids. GC/MS of seed oil extracted by ultrasound-assisted extraction showed the presence of higher content of oleic acid comparable with other extraction techniques. The petroleum ether fraction of the leaves showed the most potent hepatoprotective and anticancer effects, whereas the ethanolic extract was active against the tested gram-positive and gram-negative microorganisms. Our findings confirm that M. oleifera L. cultivated in Egypt has unique phytochemical content (comparable with M. oleifera cultivated in other countries); consecutively, it has many potent biological activities. So, it is highly recommended to cultivate the plant species on a wide scale to make use of its constituents in pharmaceutical and nutraceutical industries.
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Production of isoflavones-enriched soy yogurt through soymilk fermentation using probiotic bacteria p. 42
Asmaa I El-Shazly, Amira A Gamal, Asmaa N El-Dein, Walaa S.A Mettwally, Mohamed A Farid
DOI:10.4103/epj.epj_46_20  
Background and objective Fermented soy products were regarded as healthy foods and hence are considered an essential part of the diet. Lactic acid bacteria isolated from naturally fermented Egyptian food products were screened for their ability to produce β-glucosidase, isoflavone aglycone, phenolics, and antioxidant activity during the formation of soy yogurt. The present research is a preliminary attempt to ascertain soy yogurt production by different strains of lactic acid bacteria and their efficacy for the production of the aforementioned products. Materials and methods A total of 16 probiotic lactic acid bacteria were used for the preparation of soy yogurt and tested for their probiotic properties. Soymilk was prepared and inoculated (1% v/v) with the probiotic strains previously activated in the MRS medium. After fermentation, cell viability, pH, titratable acidity, total phenolic compounds concentration, antioxidant activity, isoflavones aglycone (daidzein and genistein), and extracellular and cell membrane-bound β-glucosidase activity were determined. Results and conclusion A total of 16 probiotic lactic acid bacteria were used for the preparation of soy yogurt. The final pH of the fermented soymilk ranged from 4.92 to 6.6, and their titratable acidity (lactic acid %) ranged from 0.5 to 0.99%. Changes in β-glucosidase, isoflavone aglycone, total phenolics content, and antioxidant activity during the formation of soy yogurt were determined. All bacterial isolates showed positive cell-bound and extracellular β-glucosidase. Their activities ranged from 308.65 to 553 mU/ml. The Lactobacillus strains showed lower extracellular than their cell-bound β-glucosidase, and the opposite was true for the other group. An increase in the content of isoflavone aglycones in soy yogurt could be achieved by aging with bacterial fermentation. Soymilk fermented with Lactobacillus strains showed the highest bioconversion of isoflavone glucosides into isoflavone aglycones than other strains, although they produced less β-glucosidase enzymes. The antioxidant activity is related to changes in total phenolics. All microorganisms were able to increase the total phenols, whereas some Lactobacillus strains were unable to release more phenols compared with unfermented soymilk.
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Statistical optimization of L-asparaginase production by Cladosporium tenuissimum p. 51
Mahdi Hamed, Ahmed A Osman, Mustafa Ateş
DOI:10.4103/epj.epj_47_20  
Background L-asparaginase produced by plant and bacteria can be used in the pharmaceutical and food industry. Unlike the bacterial counterparts, fungal L-asparaginase has more stability, more activity, and less adverse effects. Central composite design (CCD) was used to optimize temperature, pH, incubation time, and carbon-to-nitrogen ratio for L-asparaginase production by Cladosporium tenuissimum via submerged fermentation. CCD reduces the number of tests and time for optimization. Objective To optimize the culture conditions, such as temperature, pH, production time, and the ratio between concentration of carbon and nitrogen sources, for the production of L-asparaginase by isolated C. tenuissimum via submerged fermentation. Materials and methods Primarily, four significant parameters (temperature, pH, incubation period, and carbon-to-nitrogen ratio) were identified that affect the production process of L-asparaginase via submerged fermentation using the modified Czapek Dox medium. CCD was used to optimize the selected parameters concurrently, and their results were compared. Results and conclusions The highest L-asparaginase enzyme activity obtained was 2.6471 U/ml at 37°C, pH 6.2, incubation time 72 h, and 2 : 1 carbon-to-nitrogen ratio. The P value of interaction between every two factors was only significant for the interaction between temperature and incubation period (P<0.0281). The most significant factor was temperature followed by pH (P<0.0154) and carbon-to-nitrogen ratio (P<0.0346). Incubation period has no major effect on the production of L-asparaginase, but it has a quadratic effect (P<0.0001). Our results showed the significant role of culture conditions (temperature, pH, incubation period, and carbon-to-nitrogen ratio) in L-asparaginase production and confirmed the need for optimization.
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Production of a bacterial extracellular L-glutaminase possessing high antioxidant capability Highly accessed article p. 59
Sara M El-Sousy, Saadia M Easa, Amira A Hassan, Abdel-Mohsen S Ismail
DOI:10.4103/epj.epj_51_20  
Background and objectives L-glutaminase has utmost practical importance in many fields, such as medicine, pharmacy, and some industries as an effective antioxidant, anticancer, flavor enhancer, and used as an analytical reagent in the determination of glutamate and glutamine. The objective of the present article was to formulate the production medium and to pinpoint the proper growth conditions for the most potent microorganism producing highly active glutaminase enzyme. The general properties of the crude enzyme preparation were determined to detect the proper conditions for enzyme activity. Under the specified conditions, the capabilities of the crude L-glutaminase preparation for antimicrobial and antioxidant activities were investigated. Materials and methods A total of 12 recommended microbial strains were screened for highly active L-glutaminase enzyme production. Factors influencing the production of L-glutaminase enzyme were optimized, and the important properties of the crude enzyme were pinpointed. Finally, biological activities of the crude enzyme were investigated as a preliminary index for the validity of the partially purified L-glutaminase form for medical applications. Results and conclusion Among all tested microorganisms, Bacillus subtilis NRRL 1315 was the most potent producer for L-glutaminase enzyme. The maximum glutaminase production was obtained after 48 h of incubation on a rotary shaker (150 rpm) with the medium containing 5 g/l glucose, 0.1 g/l sodium nitrate, and 10 g/l L-glutamine at 37°C and pH 7.5. The important properties of the crude L-glutaminase were duly pinpointed as follows: optimum enzyme protein concentration and substrate concentration were 2 mg/ml and 40 mM, respectively, and optimum reaction pH and temperature were 7.5 and 37°C, respectively. Under the specified conditions, the crude enzyme exhibited considerable 2, 2′-diphenyl-1-picrylhydrazyl radical scavenging activity.
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Protective effect of nanoencapsulated curcumin against boldenone-induced testicular toxicity and oxidative stress in male albino rats p. 72
Mohamed A.S Aly, Marwa El-Sayed El-Shamarka, Tarek N Soliman, Mahmoud A.E Elgabry
DOI:10.4103/epj.epj_53_20  
Background Boldenone (BOL) (Equigan) is a synthetic anabolic steroid used mainly by veterinarians to treat and promote horses’ growth. Recently, body builders have started to use it to enhance their physical performance and muscle growth. Equigan is known to induce testicular injury and reduce fertility in males. Current treatments for reduced fertility are very costly. As alternatives, people are investigating naturally occurring bioactive compounds in plants such as curcumin. Objective This study was conducted to evaluate the prophylactic antioxidant effects of nanoencapsulated curcumin (NEC) on BOL-induced testicular toxicity and oxidative stress in male albino rats. Materials and methods NEC was prepared using a novel freeze-drying method. For their characterization, ultraviolet–visible spectrophotometry, transmission electron microscopy, and dynamic light scattering were used. Four groups of male rats were used: the first group served as control, the second group received NEC (100 mg/kg orally, once daily), the third group received BOL (5 mg/kg intramascular, once weekly) for 60 days, and BOL and NEC were concurrently administered in the fourth group. Blood was withdrawn from the rats’ retro-orbital veins 24 h after treatment. Animals were euthanized immediately; the epididymal sperm reserve was separated. Then, one testis from each rat was kept at −80°C for determination of oxidative stress indices, and the other was fixed in 10% formalin solution for histopathological investigation. Results and conclusion Treatment with BOL resulted in significant reproductive damage caused by increased levels of malondialdehyde and nitric oxide and decreased levels of superoxide dismutase and reduced glutathione. Downregulation of the levels of serum testosterone and reduction in semen quantity, sperm count, and motility were also detected in the BOL group. Histopathological examinations showed severe degenerative changes in the testes. Immunohistochemical examination indicated severe reduction in the proliferating cell nuclear antigen-positive spermatogonia in the BOL-treated group as compared with the control. Coadministration of NEC with BOL effectively reduces BOL-induced testicular damage and oxidative stress in male albino rats.
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ORIGINAL RESEARCH ARTICLE Top

Liquid chromatography–electro spray ionization–tandem mass spectroscopy method for the quantification of alogliptin in spiked human plasma p. 82

DOI:10.4103/epj.epj_55_20  
Background Alogliptin is the inhibitor of dipeptidyl peptidase-4. It acts to regulate blood sugar by increasing the amount of insulin in the body. Aim A liquid chromatography–mass spectroscopy/mass spectroscopy method was developed for the estimation of alogliptin in spiked human plasma. Liquid–liquid extraction technique was adopted for the extraction of alogliptin from human plasma. Materials and methods The chromatographic separation was performed on a Waters Symmetry shield RP C-18 column with 4.6-mm internal diameter, 5-μm particle size, 100A° pore size analytical column at a flow rate of 1.0 ml/min using a mixture of 0.3% formic acid and acetonitrile in the ratio of 20 : 80% v/v as mobile phase. Positive ion mode was selected to obtain the product ion m/z +339.19 (parent) →245.11 (product) for alogliptin and m/z +303.39 (parent) →232.16 (product) for internal standard. Results The developed method was satisfactorily validated as per United State Food and Drug Administration guidelines for the bioanalytical study because it exhibits excellent intraday and interday accuracy with % nominal 91.06→98.48% and precision percentage coefficient variation less than or equal to 2% in all quality control levels. Alogliptin revealed its linearity with correlation coefficient (r2=0.99) in the concentration range of 40.17–16 096 ng/ml, showed acceptable % extraction recovery (96.18→98.36%), and showed excellent matrix and analyte selectivity (% interference=0), as well as matrix effect (matrix factor 0.931 at lower quantitation limit and 1.14 at high quality control level). The stability study results of bench top, freeze thaw, autosampler, and short-term and long-term showed the accuracy in the range of 92.52–99.16% and percentage coefficient variation in the range of 0.22–1.93. Conclusion The method was successfully optimized and validated as per the United State Food and Drug Administration bioanalytical method development guidelines. The applicability of the developed method undoubtedly can further extend during preclinical and clinical trials.
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Moringa (Moringa oleifera) leaves aqueous extract enhances fibronectin type III domain-containing protein 5 gene expression and serum irisin liberation in an obesity model p. 92

DOI:10.4103/epj.epj_50_20  
Background Obesity, a risk agent for many chronic diseases, leads to increased mortality and poses one of the major public health problems. Objective This study aimed to investigate the thermogenic and antiobese efficiency of Moringa aqueous extract (MAE) on obese-modeled rats. Materials and methods Adult male rats (150–170 g) were randomly divided into four groups, with 10 animals each, as follows: (a) healthy rats served as control, (b) healthy rats administrated with MAE (400 mg/kg/day), (c) obese-modeled rats, and (d) obese-modeled rats treated with MAE. Results After 30 consecutive days of treatment, the obtained results declared that MAE possessed antiobesity, thermogenic, antilipidemic, and antiinflammatory potential. MAE succeeded significantly in reduction of the BMI and serum leptin level coupled with up-regulation of fibronectin type III domain-containing protein 5 gene mRNA expression and serum irisin level. It clearly increased serum paraoxonase-1 activity and improved lipid profile values. Moreover, it markedly reduced serum tumor necrosis factor α and increased antioxidant activity, which was achieved from the marked improvement in malondialdehyde, nitric oxide, catalase, superoxide dismutase, and glutathione values in cardio-hepatic tissues. These findings were confirmed by the regeneration of the hepatic histopathological structure. Conclusion MAE, as a food supplement, could play a beneficial role in management of obesity and restoring its complications. This could be exhibited through multiple pathways, mainly via upregulation of fibronectin type III domain-containing protein 5 gene expression and production of the soluble myokine ‘irisin,’ which is responsible for browning of white adipose tissue as well as increment of total body energy expenditure.
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