Formulation Development and Characterization of Darunavir and Ritonavir Sustained Release Tablets Using Quality by Design Approach

Darunavir is a nonpeptidic inhibitor of protease and is primarily metabolized by cytochrome P450 3A (CYP3A) isoenzymes. It is usually coadministered with low-dose ritonavir (Darunavir/r). Ritonavir is an inhibitor of CYP3A isoenzymes and pharmacologically enhances Darunavir which leads to increased plasma concentrations of darunavir and allows for daily lower dose. Here, we have developed combination SR formulation of Darunavir and Ritonavir and evaluated. In vitro drug release of all formulations was carried out in dissolution medium 900ml of pH 3.0, 0.05 M Sodium Phosphate Buffer + 2% Tween 20 for 75 RPM USP II apparatus (paddle). The results shown that, all the formulations of matrix tablets shown the good release of drug from trialed formulations however all formulations were not releasing the drug in enough amount. In matrix tablets F6, the release of drug shows NLT 80%. So, the formulation F6 have been considered as suitable for the SR tablet of Darunavir and Ritonavir. Tablets were also evaluated though Quality by Design (QbD) method.

Crosslinking Efficacy and Cytotoxicity of Genipin and Its Activated Form Prepared by Warming It in a Phosphate Buffer: A Comparative Study

The aim of the present study was to compare the acute and cumulative cytotoxicity of intact (n-GE) and warmed genipin (w-GE), while investigating the differences in crosslinking capabilities of these two genipins by rheological and mechanical tests. The n-GE solution was prepared by dissolving genipin powder in a sodium phosphate buffer solution. The w-GE solution was prepared by warming the n-GE solution at 37 °C for 24 h. The mechanical tests for chitosan (CH)/genipin gels showed the crosslinking rate of w-GE was much greater than that of n-GE up until 6 h after preparation, whereas the degree of crosslinking of CH/n-GE gels became higher at 12 h. The ISO 10993-5 standard method, which is established specifically for evaluating cumulative cytotoxicity, determined equivalent IC50 for w-GE (0.173 mM) and n-GE (0.166 mM). On the other hand, custom-made cytotoxicity tests using a WST-8 assay after 1 h of cultivation showed that the acute cytotoxicity of w-GE was significantly higher than that of n-GE at concentrations between 0.1–5 mM. The acute cytotoxicity of w-GE should be taken into consideration in its practical uses, despite the fact that the much faster crosslinking of w-GE is useful as an effective cross linker for in-situ forming gels.

Extracting Sweetening and Bioactive Compounds From Stevia Rebaudiana Using Cellulase Enzyme

This study was conducted to find out the sweetener and bioactive compounds in the Stevia plant’s enzyme extract and its antioxidant and antibacterial effect. The enzyme extract was used in a ratio of 1:15 (w: v) with the use of sodium phosphate buffer solution (pH=4) in an equal mixing ratio with the enzyme. Extraction was carried out at 55°C with a time of 25 minutes to extract and determine steviol glycosides using HPLC. Stevioside and Rebaudioside-A (5.101 and 3.027 mg/g) were obtained, respectively. The study showed that Stevia contains high amounts of phenols and flavonoids (83.052 and 71,765) mg/ml, respectively. The enzyme extract gave an antioxidant activity of 71.367% compared to BHT which gave an activity of 77.267%. It gave the highest inhibitory activity against Bacillus subtilis, which was 14 mm, followed by Staphylococcus aureus and E. coli. The bioactive compounds were diagnosed by GC-MS and contained important bioactive compounds such as Hydroxydehydrostevic acid (steviol), 1,2-Benzenediol, 4-Ethylcatechol, 9-Octadecenamide, (Z)-, Isosteviol methyl ester, gamma-Sitosterol.

Formulation Development and Characterization of Darunavir and Ritonavir Sustained Release Tablets using Quality by Design Approach

Darunavir is a nonpeptidic inhibitor of protease and is primarily metabolized by cytochrome P450 3A (CYP3A) isoenzymes. It is usually coadministered with low-dose ritonavir (Darunavir/r). Ritonavir is an inhibitor of CYP3A isoenzymes and pharmacologically enhances Darunavir which leads to increased plasma concentrations of darunavir and allows for daily lower dose. Here, we have developed combination SR formulation of Darunavir and Ritonavir and evaluated. In vitro drug release of all formulations was carried out in dissolution medium 900ml of pH 3.0, 0.05 M Sodium Phosphate Buffer + 2% Tween 20 for 75 RPM USP II apparatus (paddle). The results shown that, all the formulations of matrix tablets shown the good release of drug from trialed formulations however all formulations were not releasing the drug in enough amount. In matrix tablets F6, the release of drug shows NLT 80%. So, the formulation F6 have been considered as suitable for the SR tablet of Darunavir and Ritonavir. Tablets were also evaluated though Quality by Design (QbD) method.

Evaluation of the ameliorative roles of vitamins A, C and E on reduced glutathione in Clarias gariepinus (Burchell, 1822) fingerlings exposed to lead nitrate

The ever-increasing anthropogenic activities all over the world that usually led to release of plethora of pollutants such as lead calls for concern. In the present study the effects of lead nitrate on the production of antioxidants such as reduced glutathione (GSH) in C. gariepinus and how such effects can be ameliorated through administration of vitamins were investigated. C. gariepinus fingerlings (whose initial weight ranged from 3-11g) were exposed to sub-lethal concentrations of Pb (00, 26mg/L, 44mg/L, 61mg/L and 79mg/L) with replicate in each case. 26mg/L each of the vitamins were administered across all bud. Fresh concentrations of both toxicant and vitamins were administered every 72 hours for a period of 12 weeks every time the water medium was changed. The various treatments group include Pb (Pb only), PbVA (Pb+vitamin A), PbVC ((Pb+vitamin C) and PbVE (Pb+vitamin E) with T1-T4 and replicates in each case. 3 samples of the fish were randomly selected and sacrificed from each aquarium tank every 2 weeks of the exposure period. The gills, kidneys and liver were excised from these specimens and homogenized in sodium phosphate buffer. These were then assayed for GSH productions levels in each case. The data generated were subjected to one way analysis of variance and considered significant at P≤0.05. From the results: In the Pb only group, the mean values of the GSH produced in the liver of the control samples were significantly higher than other treatments. The highest mean values of 82.04±0.13µg/ml, 30.84±0.10µg/ml and 31.30±0.10µg/ml were obtained in the liver, kidney and gills of the fish, respectively. In fish samples exposed to PbVA group, the highest mean values of 23.57±0.10µg/ml, 58.74±0.07µg/ml and 52.72±0.07µg/ml were obtained in the liver, kidney and gills, respectively. In C. gariepinus exposed to PbVC group, the highest mean values obtained in the liver, kidneys and gills were 25.79±0.07µg/ml, 28.40±0.13µg/ml and 37.55±0.03µg/ml, respectively. In PbVE group, the highest mean value of 57.21±0.03µg/ml, 83.51±0.07µg/ml and 63.29±0.07µg/ml were btained in the liver, kidneys and gills, respectively. The liver of the samples exposed to Pb only group displayed higher level of response to the toxicant with the highest GSH produced in the lowest concentration in comparison to other fish organs. In the PbVA group the response was more in the kidney in the highest concentration. There were general low levels of production in all organs of the fish in the PbVC group. The kidneys of the PbVE group exhibited the highest level of GSH production in comparison to other organs. The kidneys and liver of C. gariepinus in this research were fully engaged in mitigating the effects of the toxicant in the presence of the vitamins. Administration of higher concentrations of the vitamins could enhance better understanding of the ameliorative roles of the vitamins against the deleterious effects of the toxicant.

Evaluation of the ameliorative roles of Vitamins A, C and E on reduced glutathione in Clarias gariepinus (Burchell, 1822) fingerlings exposed to cadmium chloride

Effects of cadmium chloride on the production of antioxidants such as reduced glutathione (GSH) in Clarias gariepinus and how such effects can be ameliorated through administration of vitamins were investigated. C. gariepinus fingerlings were exposed to sub-lethal concentrations of Cd (00, 12mg/L, 16mg/L, 20mg/L and 24mg/L) with replicate in each case. 12mg/L each of the vitamins were administered across all bud. Fresh concentrations of both toxicant and vitamins were administered every 72 hours for a period of 12 weeks every time the water medium was changed. 3 samples of the fish were randomly selected and sacrificed from each aquarium tank every 2 weeks. The gills, kidneys and liver were excised from these specimens, homogenized in sodium phosphate buffer and then assayed for GSH production levels in each case. From the results: In Cd only group, the highest GSH level produced in the liver was 38.85±0.07µg/ml. In the liver of samples of CdVA group, the value (93.97±0.07µg/ml) increased then followed by the gill (67.72±0.13µg/ml). In CdVC, the GSH production level in the gill (39.76±0.07µg/ml) was relatively higher than livers and kidneys of the samples. In CdVE, the kidney produced the highest GSH value of 32.89±0.10µg/ml. The elicitation and utilization of the antioxidant at one point or the other were adopted by the fish in dealing with the effects of the toxicant especially in the presence of the vitamins. Higher concentrations of the vitamins could facilitate the understanding of the effects of the vitamins in mitigating the effects of the toxicant.

A method for the temperature-controlled extraction of DNA from ancient bones

Contamination with microbial and other exogenous DNA poses a significant challenge in the generation of genome-wide sequence data from ancient skeletal remains. Here we describe a method for separating ancient DNA into multiple fractions during DNA extraction by sequential temperature-controlled release of DNA into sodium phosphate buffer. An evaluation of the effectiveness of the method using a set of three ancient bones resulted in between 1.6- and 32-fold enrichment of endogenous DNA compared with regular DNA extraction. For two bones, the method outperformed previous methods of decontaminating ancient bones, including hypochlorite treatment, which resulted in near-complete destruction of DNA in the worst-preserved sample. This extraction method expands the spectrum of methods available for depleting contaminant DNA from ancient skeletal remains.

OPTIMIZATION OF THE WASTE PROCESSING PROCEDURE OF THE BEER INDUSTRY AND OBTAINING LIPID PREPARATION FROM YEAST BIOMASS

Currently, special attention is paid to the use of industrial by-products, in particular those obtained in huge quantities in the brewing and wine making process, as a source for the production of natural preparations with high biological value. In this study are presented results related to the recovery of beer yeast biomass from the sediments of the beer industry, by obtaining lipid extracts with valuable biochemical composition. Thus, applying the optimized autolysis method, with the use of sodium phosphate buffer at 45°C for the destruction of the cell wall and fractional extraction with hydric, alkaline and acidic solutions, various liquid and solid fractions were obtained from the biomass of brewer’s yeast with varied lipid content. So, in order to optimize the waste processing process of the beer industry and the complex recovery of yeast biomass, it is reasonable to include the lipid fraction extraction stage in the technological flow, after obtaining protein and mannoprotein extracts, which will be useful and as an additional step of purification of the β-glucan fraction.

α-amylase from white pitaya (Hylocereus undatus L.) peel: optimization of extraction using full factorial design

Introduction. Amylase is a significant enzyme with numerous commercial applications, which is largely used to convert starches into oligosaccharides. Extraction of amylase from plant by-products or cheap sources is cost-effective. Annually, pitaya fruit juice industry produces huge amounts of peels that could be utilized as an alternative source in enzyme production industry. The work aimed to examine and optimize extraction process. Study objects and methods. In this study, we investigated parameters of extraction to optimize the process, as well as activity of α-amylase from white pitaya fruit (Hylocereus undatus L.) peel. For this purpose, a two-level full factorial design was applied. Three variables, namely the pH of sodium phosphate buffer (X1, 4.5–7.5), mixing time (X2, 1–3 min), and a sample-to-buffer ratio (X3, 1:3–1:5), were used to identify significant effects and interactions within the samples. Results and discussion. The results demonstrated that the buffer pH had the most significant (P ≤ 0.05) effect on total amylase activity. Based on full factorial design analysis, we revealed the optimal conditions for amylase enzyme extraction ‒ pH of 6, mixing time of 2 min, and a sample-to-buffer ratio of 1:4. Lower and higher values influenced adversely on specific activity of amylase. Conclusion. Optimization increased the enzyme specific activity by a factor of 4.5. Thus, pitaya peel could be used in different industries as a rich natural α-amylase source.

The occurrence of strawberry virus 1 infecting strawberry in Shandong province, China

Strawberry (Fragaria × ananassa Duch.) is one of the most important horticultural plants worldwide with high economic and nutritional value. Strawberry associated virus 1 (SaV1) is a putative Cytorhabdovirus isolated from strawberry in Fujian province, China (Ding et al., 2019). Strawberry virus 1 (StrV-1) is another putative Cytorhabdovirus characterized from F. ananassa and F. vesca in Czech Republic (Fránová et al., 2019). The complete genomes of isolates of SaV1 and StrV-1 share 79 to 98% nucleotide (nt) identities. In August 2020, foliar chlorotic spots or streaks were observed in four strawberry cultivars (cv. Honeoye, Mibao, 8128 and All Star) in Yantai, Shandong province, China. To identify the associated viruses, symptomatic leaves from two plants of each cultivar (8 samples) were pooled for high-throughput sequencing (HTS). Total RNA was extracted from the composite sample and used for constructing a cDNA library after ribosomal RNA (rRNA)-depletion. Sequencing was carried out on Illumina Hiseq 4000 (Novogene, China). Raw reads were filtered, trimmed and de novo assembled as described previously (Grabherr et al., 2013; Zhou et al. 2020). The resulting contigs were screened by BLASTn and BLASTx against GenBank database. Subsequent analyses indicated the presence of strawberry vein banding virus, strawberry pallidosis associated virus and strawberry mottle virus in the analyzed sample, which had been reported previously in strawberry (Martin and Tzanetakis, 2013; Shi et al., 2018; Bhagwat et al., 2016). Besides, five contigs ranging from 266 to 6,057 nt were obtained. They shared 87 to 91% nt sequence identity with StrV-1 isolate B (GenBank accession no. MK211271). To confirm StrV-1 infection in the strawberry plants, total RNA was isolated from all eight samples using RNAprep Pure Plant Plus Kit (Tiangen, China). Reverse transcription polymerase chain reaction (RT-PCR) was conducted with two pairs of specific primers StrVp1 (Forward: 5ʹ-CATTACTGAAGCATTCCGTG-3′/Reverse: 5ʹ-AGATATCACGCACAGTGAC-3ʹ), and StrVp2 (Forward: 5ʹ-TTGCGCGAAGCGGATGTCCG-3′/Reverse: 5ʹ-GGCTGCCAGAGCGTTGGATG-3ʹ), targeting nt positions 70-1,231 and 7,825-9,348 of StrV-1 isolate B, respectively. Fragments with the expected sizes were amplified from two samples of cv. All Star. The amplicons were cloned, sequenced, and deposited in GenBank under accession no. MW419123-124 and MW645247-248. Both protein encoding sequences shared 91 to 92% and 80 to 84% nt identities with the corresponding sequences of StrV-1 isolate B and SaV1, respectively, indicating that the isolates from this study are genetic variants of StrV-1 and distantly related to SaV1. Crude sap was prepared by homogenizing leaf tissues of StrV-1 infected strawberry in 0.02 mol/L sodium phosphate buffer with 0.45% (w/v) sodium diethyldithiocarbamate thihydrate, then gently rubbed onto five healthy Nicotiana benthamiana plants. Neither the inoculated leaves nor the systemically infected leaves showed obvious symptoms seven days post inoculation. However, StrV-1 was detected by RT-PCR in all five N. benthamiana plants as described above. In addition, a survey of strawberry greenhouses was conducted in August 2020 and approximately 10% of plants in a 667 m2 greenhouse in Yantai had StrV-1-like symptoms. To the best of our knowledge, this is the first report of the occurrence of StrV-1 infecting strawberry in Shandong province, China. Our findings expand the geographic range and genetic diversity of StrV-1 and indicate it could be a potential virus threat to strawberry production in China.