The biochemical and growth-associated traits of basil (Ocimum basilicum L.) affected by silver nanoparticles and silver.

BACKGROUND: The biochemical and growth changes resulting from exposure of basil (Ocimum basilicum L.) seedlings to silver nanoparticles and silver were investigated. Over a two-week period, seedlings were exposed to different concentrations (0, 40, and 80 ppm) of silver nanoparticles and silver. RESULTS: Our findings revealed that at concentrations of 40 and 80 ppm, both silver nanoparticles and silver nitrate led to decreased weight, root and shoot length, as well as chlorophyll a and b content. Conversely, these treatments triggered an increase in key biochemical properties, such as total phenols, carotenoids and anthocyanins, with silver nanoparticles showing a more pronounced effect compared to silver nitrate. Moreover, the levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) rose proportionally with treatment concentration, with the nanoparticle treatment exhibiting a more substantial increase. Silver content showed a significant upswing in both roots and leaves as treatment concentrations increased. CONCLUSIONS: Application of varying concentrations of silver nanoparticles and silver nitrate on basil plants resulted in reduced growth and lower chlorophyll content, while simultaneously boosting the production of antioxidant compounds. Notably, anthocyanin, carotenoid, and total phenol increased significantly. However, despite this increase in antioxidant activity, the plant remained unable to fully mitigate the oxidative stress induced by silver and silver nanoparticles.

Carnauba wax-based edible coatings retain quality enhancement of orange (Citrus sinensis cv. Moro) fruits during storage.

Fruit coatings serve a dual purpose in preserving the quality of fruits. Not only do they act as a barrier against water evaporation and fungal infiltration, but they also enhance the fruit's visual appeal in the market. Yet, their influence on the fruit's quality components, which play a crucial role in determining its nutritional value, taste, and overall flavor, has remained relatively unexplored. This study aimed to evaluate the effects of carnauba wax coating on the quality of Moro oranges during storage. The selected fruits were meticulously chosen for uniformity in size. The experiment involved applying carnauba wax, a commonly used type among local producers, at four different concentrations: 0%, 0.5%, 1%, and 1.5%. These treatments were applied during various storage periods, including immediately after fruits were harvested and after 40 and 80 days. Following the application of these treatments, the oranges were stored in a controlled environment (morgue) at a temperature of 4 ± 1 °C. Subsequently, several physicochemical parameters of both the fruit flesh and skin were examined. The results unveiled a decline in the overall ascorbic acid content of the fruits. In terms of phenol content, a general decreasing trend was observed after harvesting. At each sampling interval during storage, the phenol content in uncoated fruits consistently exceeded that of their waxed counterparts. Significant reduction in fruit weight was observed throughout the storage period. Both vitamin C and total acidity levels in the fruit exhibited decreases during the storage period. As time passed, fruit firmness gradually declined, while fruit decay increased during the 40- and 80-day storage periods for untreated Moro oranges. The anthocyanin content showed an increasing trend. The study also unveiled a decline in the antioxidant capacity of citrus fruits during storage. Strong significant positive correlations were observed between total phenol content and key parameters, such as antioxidant activity (0.941**), MDA (0.364*), vitamin C content, and total carbohydrate content (0.475**). Skin radiance showed a perfect correlation with chroma and hue (1.000**). Principal component analysis revealed that the first principal component accounted for 34.27% of the total variance, out of a total of five principal components that explained 77.14% of the variance. Through cluster analysis, the variables were categorized into three distinct groups; one associated with weight loss and another with ion leakage. Considering these findings, carnauba wax-based coating emerges as a promising solution for preserving Moro oranges. It effectively mitigates fruit weight loss and helps maintain fruit firmness during storage, making it a valuable tool for fruit preservation.

Severity and injury characteristics among matched hospitalized motorcycle drivers and their passengers.

PURPOSE: After car accident, motorcycle accident ranks as the second leading cause of traffic fatality in Iran. This study aimed to compare the severity and clinical presentations between drivers and passengers under the same injury circumstance. METHODS: This study was conducted in the trauma center of Shiraz, Iran in 2017. Data on demographics, triage level, blood pressure, respiratory rate, Glasgow coma scale (GCS), injured body region, injury severity score (ISS), revised trauma score (RTS), and result of accident were compared between pairs of drivers and passengers. The agreement of any type of injury between drivers and passengers evaluated by Kappa test. RESULTS: This study included 143 matched pairs of drivers and passengers. Most of the pairs (84.5%) did not use helmet and 77.2% of the riders do not have driving license. ISS was significantly higher in drivers than passengers. In the unmatched pairs, drivers and passengers showed no difference in sustaining injuries in the face, head & neck, chest and soft tissue, but drivers were found more likely to suffer from injuries in the abdomen, extremities, pelvis and spine than passengers. Once one part of the matched pair suffered injury in the head & neck, face, chest, abdomen, extremities and soft tissue & skin injury, the probability that the other part had an injury in the same region was 50%, 9%, 13%, 7%, 22% and 34% respectively. Kappa value for these body regions was 0.006, 0.009, -0.006, 0.068, 0.063 and 0.001, respectively, which was significant in abdomen and extremities. CONCLUSION: Although drivers had higher level of injury severity and some different injury distributions, we recommend equal treatment to drivers and passengers. We also recommend related authorities to develop policies on helmet use, driving license and third-party insurance.

Selective high throughput protein quantification based on UV absorption spectra.

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Recently, a new label-free and non-invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements was presented. Analytics based on the methodology was shown to increase analytical throughput for selective protein quantification significantly, however this was only demonstrated for one particular protein combination. In this work, the possibilities and limitations of the analytical method are investigated further. Principal component analysis (PCA) was performed on a broad range of absorption spectra to investigate their common characteristics and differences. The PCA was used both for cluster analysis and to define a measure for spectral similarity. For binary protein combinations, the calibration precision was shown to decrease exponentially with the defined spectral similarity factor. Knowledge of this correlation can be used to determine a priori whether a calibration will be successful or not. Calibration robustness was investigated by applying the analytics to liquid chromatography performed in HTE mode. Further it was shown, that a spectral difference of 0.6% was sufficient to sucessfully preform a spectral based calibration of two IgG1 monoclonals.

Investigation of a dual CD chiral CE system for separation of glitazone compounds.

A dual CD-CE method for chiral separation of enantiomers of pioglitazone, rosiglitazone and balaglitazone was investigated for the purpose of optimizing the chiral separation. In a previous work a dual CD chiral CE method was used for investigation of glitazone compounds in drug substance and pharmaceutical formulation and the studies showed that all studied glitazones were racemic mixtures. This CE method could separate the enantiomers with a resolution (R(S)) of about 3. However, another study on single glitazone enantiomers pointed out that a higher R(S) is needed to achieve more accurate results for separation of a small amount of one enantiomer in the presence of a high amount of the other enantiomers. The focus of this investigation was thus directed toward the effect of CDs and the pH of the running buffer to achieve a better enantioseparation. Initially CE systems with each of heptakis(2,6-di-O-methyl)-beta-CD (DM-beta-CD) and heptakis(6-sulfobutylether)-beta-CD (SB-beta-CD) as single CD added were investigated at three different pH values (2.5, 5.0 and 9.3). After having chosen the best of these three pH values a dual CD system was further investigated and optimized. The optimization work was then focused on the concentration of the two CDs and the pH of the running buffer and was performed using factorial design experiments. A mixture of a DM-beta-CD and SB-beta-CD was found to be optimal and necessary to achieve enantioseparation with sufficiently high R(S). In order to further verify the importance of the SB-beta-CD, a CE system with the DM-beta-CD added and substitution or partial substitution of the SB-beta-CD by SDS was studied for comparison. (1)H-NMR studies were performed to get a more detailed understanding of the interactions between the glitazones and the CDs used.The optimized dual CD-CE method for chiral separation of the enantiomers of pioglitazone, rosiglitazone and balaglitazone using a running buffer containing 50 mM borate buffer pH 9.7, 12 mM of SB-beta-CD and 3 mM of DM-beta-CD provided a high R(S) (R(S) between 5.5 and 8.8).

Investigation of racemisation of the enantiomers of glitazone drug compounds at different pH using chiral HPLC and chiral CE.

Drug enantiomers can have biologically distinct interactions within the biological system and consequently different pharmacological or toxicological effects. Development of a better and safer drug product may be considered if one of the enantiomers has a significantly better effect/side effect ratio than the other. Investigation of the single enantiomers in a racemic mixture could be valuable in order to investigate whether the single enantiomers demonstrate difference in pharmacological effect and/or fewer side effects versus the racemic mixture. In this context investigation of a possible racemisation of the pure enantiomers is very important. In order to obtain the enantiomers of the racemic pioglitazone and the racemic rosiglitazone an HPLC method for chiral separation was developed. Using this method the R and S enantiomers were separated and the method was used to collect each enantiomer for investigation of racemisation process. The racemisation of the enantiomers of pioglitazone and rosiglitazone was investigated at pH 2.5, 7.4 and 9.3 using a chiral CE system. At pH 2.5 all enantiomers showed a slow racemisation. After 192 h (8 days) at 37 degrees C the ratio of the enantiomers in the mixture for all four isolated enantiomers was approximately 2 to 1 and after 1440 h (30 days) full racemisation was observed. The racemisation speed increased with increasing pH. At pH 7.4 the ratio of the enantiomers in the mixtures was approximately 2 to 1 already after 10h. Full racemisation was observed within 48 h (2 days) at pH 7.4 and within 24 h at pH 9.3. These investigations have shown that it is possible to separate and isolate the enantiomers from a racemic mixture of glitazone drug substance and perform racemisation studies on each enantiomer.

Generic, highly selective and robust capillary electrophoresis method for separation of a racemic mixture of glitazone compounds.

A generic, highly selective, and robust capillary electrophoresis (CE) method was developed for separation of a racemic mixture of three available glitazone compounds (also known as thiazolidinediones) in active pharmaceutical ingredients (API) and tablets. The method separated the R and S enantiomers of balaglitazone, pioglitazone and rosiglitazone, and showed that the samples contained an equal (50:50) quantity of the enantiomers as a mixture. After a simple extraction of samples with acetonitrile:water (80:20), separation was performed using a combination of two cyclodextrins: sulfobuthylether-beta-cyclodextrin (SB-beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) in the electrolyte at pH 8.0. The method showed a very good specificity, and all separations were achieved with a resolution (Rs) over 3.0. The developed CE method was then validated. The Rs for the separations were 3.5 for balaglitazone enantiomers, 3.5 for pioglitazone enantiomers, and 3.7 for rosiglitazone. The squared correlation coefficients (r2) were found to be 0.999 for all three compounds. The range of the CE method (injection volume was approximately 4 nl) was demonstrated to be from 1.0 to 2.4 ng. The R.S.D. in the repeatability study was found to be less than 0.5 for area/area ratio (and 3.0% for area) for all three compounds. The R.S.D. in the intermediate precision study was found to be less than 0.7 for area/area ratio (and 4.5% for area) for all three compounds. Generally, the method showed good robustness. Resolution between the enantiomers peak was maintained acceptable throughout the small variations around the pH value of the buffer, different capillary, CE instrument and electrolytes ion strength capacity, but changes in concentration of cyclodextrins and acetonitrile showed significant effects on separations and affected the resolution. The validation results showed that the CE method was suitable for separation of the racemic mixtures of the three glitazone drugs. The CE method was then applied for routine test during the drug and formulation development work of balaglitazone. Due to the achieved results from this work, it is the authors' belief that this method can easily separate other glitazone racemic mixtures.

Development and validation of a method for the purity determination of (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol(FF-MAS) in pharmaceutical products containing recombinant human albumin.

A simple and sensitive method has been developed and validated for purity determination of FF-MAS (also known as (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol an endogenous substance usually present in the pre-ovulatory follicular fluid) at very low concentrations (200 ng per unit) in pharmaceutical formulations containing RECOMBUMIN (recombinant human albumin) as the matrix. The paper focuses on development of the sample preparation for the product containing recombinant human albumin. After removal of recombinant human albumin by precipitation using a mixture of water and ethanol, the FF-MAS was concentrated by evaporation using a vacuum centrifuge and the prepared sample was analyzed. The purity method was based on a reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet absorption detection at 250 nm. The method was validated according to ICH guidelines. The method indicated a significant degree of specificity with good selectivity and no significant effect from the matrix. The limit of detection was found to be 0.3-0.8% (depending on the impurity) corresponding to 1.9-5.1 ng. The limit of quantification was found to be 0.8-2.5% (depending on the impurity) corresponding to 5.2-16 ng. The recovery was found to be between 90 and 101% for the FF-MAS, and 100-129% for the six known impurities. The tested range for FF-MAS was from 320 to 960 ng corresponding to 50-150% of the nominal concentration (640 ng, injection volume is 100 microl). The linearity of each compound (FF-MAS and the six impurities) was investigated. The squared correlation coefficient (r(2)) was 0.999 for FF-MAS (50-150% level) and 0.977-0.998 for the six known impurities (at four levels: 0.20, 0.50, 1.00, 2.00%). The R.S.D. in the repeatability study was found to be 9.2% for the total amount of impurities, and 10.4% for single impurities. The R.S.D. in the intermediate precision study was found to be 10.9% for total impurities, and 12.0% for single impurities. The validation results showed that the method was suitable for the purity analysis. The validated method was then ready for use for samples analysis of phase II clinical studies and the stability investigations of the pharmaceutical product.

Development and validation of a high-resolution capillary electrophoresis method for multi-analysis of ragaglitazar and arginine in active pharmaceutical ingredients and low-dose tablets.

A selective, sensitive and robust capillary electrophoresis (CE) method has been developed and validated for multi analysis of ragaglitazar (also known as NNC 61-0029 or DRF 2725) and its counter ion arginine in Active Pharmaceutical Ingredients (API) and low-dose tablets (0.5, 1.0 and 2.0 mg). The method covers a total number of 12 analyses for the API and tablets: assay and identification of ragaglitazar and arginine, chiral purity of ragaglitazar and purity of ragaglitazar. After a simple extraction of samples with acetonitrile and 0.01 M sodium hydroxide (10:90), separation was performed using a combination of two cyclodextrins; sulfobutylether-beta-cyclodextrin (SB-beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) in the electrolyte. The method showed good specificity and could separate and detect ragaglitazar, the distomer (the (+)-enantiomer) and arginine. The LOQ was found to be 0.10%, corresponding to 0.2 ng (0.5 microg/ml) for ragaglitazar and the distomer. Linearity was observed to be from 0.5 to 15 microg/ml (range 0.2-60 ng) and 400-600 microg/ml (range 1603-2404 ng) for ragaglitazar and 166-250 microg/ml (range 668-1000 ng) for arginine. The accuracy (as percent recovery) for ragaglitazar was found to be 101-106% (at 400-600 microg/ml), 101-125% (at 0.5-15 microg/ml) and for arginine 97-101% (at 166-250 microg/ml). The repeatability for the detection of peaks as R.S.D. was found to be as follows, ragaglitazar: 0.05%, distomer: 1.01%, largest single impurity: 5.84%, total impurities: 0.90% and arginine: 2.00%. The intermediate precision for the detection of peaks as R.S.D. was found to be as follows, ragaglitazar: 0.63%, distomer: 1.98%, largest single impurity: 5.22%, total impurities: 13.17% and arginine: 3.50.

Development and validation of a capillary electrophoresis-indirect photometric detection method for the determination of the non-UV-absorbing 1,4-dideoxy-1,4-imino-D-arabinitol in active pharmaceutical ingredients, solutions and tablets using an internal standard.

A high speed, selective, and robust capillary electrophoresis (CE) method with high capacity was developed and validated for determination of assay of 1,4-dideoxy-1,4-imino-D-arabinitol in active pharmaceutical ingredients, solutions, and tablets during the development work at preclinical and Phase I and II clinical studies. 1,4-Dideoxy-1,4-imino-D-arabinitol, tartrate has (almost) no UV absorption. Therefore, the developed CE method for quantification was based on indirect UV detection. A cation CE principle was chosen using an electrolyte at pH 4.0 containing dimethyldiphenylphosphonium hydroxide, which has a strong UV absorbance. The quantification was performed using internal standard technique, by which piperidine was used as internal standard. The method was validated. The validation results showed that the CE method was suitable for the assay (and dissolution) analysis.