The overall aims of this project were to assess the pharmaceutical quality and consistency of commercially available solid dose Sutherlandia frutescens containing products (viz. tablets & capsules) by exploring the use of monitoring the pharmaceutical presentation, flavonoid profile and antioxidant activity levels and to develop/or adapt methods and specifications that may be used for the quality control of such products.Stability tests were conducted on all of the selected SCP. The products were stored under elevated temperatures and environmental humidity conditions and total phenol, antioxidant and chromatographic analysis was conducted on these samples. Samples of each of the SCP were hydrolyzed using HCL and then analyzed using HPLC to test the stability of the flavonoids present in each product. The SCP investigated in this study physically appeared to be of quite good “pharmaceutical” quality, but generally lacked information on the date of manufacture and lacked package inserts, or when these were present they contained insufficient information.Based on the results obtained, it is recommended that, the manufacturers of SCP pay more attention to the information provided on the package inserts and the storage conditions for their products. Further the levels of antioxidant activity, total phenols and flavonoid (sutherlandins A to D) be used as specifications to control the quality of commercially available solid dose Sutherlandia frutescens containing preparations on an individual basis.

The objective for this master’s thesis was to test and evaluate two methods for determining the content of tannin in a pinebarkextract. The methods used at Södra for this previously have not been specific enough, only the amount of polyphenolics have been measured. One of these methods is a test based on the Stiasny test and it determines the extracts ability to form a gel with formaldehyde. When this test was carried out it showed that the extract did not contain much tannin. The other method used at Södra measures the amount of polyphenolic substances with a spectrophotometer. The result from this method showed that the content were 50 %.   One of the two new methods that were evaluated determines the amount tannin present in the extract because of tannins ability to form a complex with proteins. According to this method, the amount tannin in the extract were 42,5 %, and 19,4 % of this in the form of tannic acid. These results are credible on basis of previous information about the extract.   The other method uses a RP- HPLC where ellagic and gallic acid were used as standards. The amount ellagic acid in the extract was determined to 0,06 %, but the result from gallic acid was inconclusive. Further analysis is necessary to evaluate the obtained results and the methods reliability.   If the metods is to be used on a regular basis, my recommendation would be to start evaluating the protein-binding method because this would be easier and less time-consuming than modifying the HPLC- method. The HPLC- method could on the other hand provide useful information about the extract, not only the amount tannin could be measured, but also which kind of tannin could be investigated.

Yellow pigment (YP) concentration is an important quality trait in durum wheat (<i>Triticum turgidum</i> L. var <i>durum</i>) and is comprised primarily of carotenoids. The main objective of our study was to measure the accumulation of carotenoids during the grain fill period to improve our understanding of the physiological basis for differences among durum wheat cultivars. Thirteen cultivars and breeding genotypes with large variation in total YP concentration (<6 µg g-1 to >15 µg g-1) were studied. Spikes were sampled from replicated field plots in 2007 and 2008 near Saskatoon and Swift Current, Saskatchewan, Canada, at 14, 21, 28 and 35 days after heading (DAH). The remainder of each plot was combined at grain maturity for YP and carotenoid analysis. Carotenoids were extracted with 1:1 methanol:dichloromethane (0.1% BHT) and quantified with HPLC. <i>Trans</i> (E)-lutein was the predominant carotenoid at maturity and was detected at 14 DAH in all genotypes. The rate and duration of E-lutein accumulation was variable among genotypes expressing high, intermediate and low YP. The accumulation of all carotenoids was lowest in genotypes expressing low YP, and suggests rate limitations early in the carotenoid biosynthetic pathway. E-zeaxanthin concentrations were highest in mature grain, but no significant differences were detected among genotypes. However, the ratio of E-zeaxanthin to E-lutein was inversely correlated with total YP, suggesting that the â,å branch of lycopene cyclization is favoured over the â,â branch in high-YP genotypes. These results provide insights to the regulation of the carotenoid biosynthetic pathway during grain fill stage in durum wheat and will facilitate breeding for higher carotenoid concentration.

Organic acids, including L- and D-lactate, explain most but not the entire elevated anion gap in diarrhea-associated metabolic acidosis. Also, D-lactate has been implicated in the neurological symptoms associated with this condition. Less-common organic compounds may influence the anion gap and neurological symptoms. This research aimed to characterize and attempt to identify a previously unidentified compound, Compound X, first noted in diarrheic acidotic calves with elevated anion gap (Omole, 1999).<p> High performance liquid chromatography (HPLC) was used to measure Compound X in biological fluids from diarrheic and healthy calves; diarrheic piglets, foals, and human infants; and calves experimentally infused with saline or acid. Attempts were made to identify Compound X using HPLC with tandem and Fourier-transform mass spectrometry.<p> Compound X was significantly higher in diarrheic calf serum (p<0.001) and lower in feces (p<0.001) and rumen fluid (p<0.001) than those fluids from healthy calves. Compound X in serum from acid-infused calves (median peak area ratio = 1.5 1.9) was lower than that of diarrheic calves (median = 4.8) and only slightly greater than that of healthy calves (median = 1.2). Serum Compound X correlated with serum D-lactate in diarrheic and healthy calves combined; however, no such correlation was observed in acid-infused calves. Conversely, a relationship between Compound X and neurological disturbance was present in acid-infused calves, but not in diarrheic calves. In other species, Compound X was highest in diarrheic infants and lowest in diarrheic piglets. Although mass spectrometry and database library searches revealed several compounds as putative matches for Compound X, none of the compounds made sense within the context of acidosis and mammalian biological fluids. Therefore, the identity of Compound X remains unknown.<p> Compound X has been established as a ubiquitous compound(s) present in the biological fluids of mammals. Compound X may be a normal intestinal compound or bacterial metabolite that crosses the intestinal epithelium during diarrhea. In spite of this, Compound X was associated with the neurological manifestations of D-lactic acidosis. Compound X`s identity was not determined, and some reasons for this and future directions are discussed.

No description available.

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

Hemoglobin is an oxygen binding protein in erythrocytes. Hemoglobin is composed of four polypeptide chains. During the fetal stage the type of hemoglobin called fetal hemoglobin (HbF) dominates. After birth HbF is replaced by adult hemoglobin (HbA). HbF persists in concentrations less than 1%. Elevated concentration of HbF in adults exists in different conditions, Talassemi for example. When the uterus is damaged and the fetus doesn’t feel well its blood can pass the placenta barrier and enter the blood stream of the mother. A venous blood sample from the mother is analyzed to determine the status of the fetus. Laboratory Medicine Västernorrland already has two methods for analyzing HbF, one routine and one on call. The routine method needed to be replaced and the possibility to use flow cytometry was investigated. In this study, results from flow cytometry using Fetal Cell Count™ kit was compared to the results from the presently used methods, Kleihauer-Betke and HPLC. Cord blood was diluted with venous blood from an adult with the same blood group in various concentrations. A number of tests were performed and showed a fairly good correlation between the different methods. However more tests will be necessary to draw any clear conclusion.

The objective of this work was the investigation of nanofiltration as a potential avenue to fractionate protein hydrolysates and produce protein hydrolysate fractions with stimulating bioactivity for the development of a supplement for a serum-free media. Mammalian cell culture is widely used for the production of therapeutic proteins such as antibodies, interleukins, and vaccines because of the ability of mammalian cells to glycosylate proteins. A complex media with the addition of serum is often required to meet the requirements of the cells. Although serum is a supplement that provides different proteins such as growth factors and hormones, serum has several disadvantages such as high cost, difficulty of downstream processing due to its high protein content and the possibility of microbiological contamination. Protein hydrolysates from plant, animal, or yeast cells contain a complex mixture of peptides and amino acids and have been shown to enhance growth of certain mammalian cell lines cultured in serum-free media. To fractionate peptide mixtures, nanofiltration was investigated in this study. Nanofiltration is a pressure driven membrane separation process based on size and charge. The investigation of pH and NaCl on the filtration performance for two different nanofiltration membranes (HL membrane and G-10 membrane) was achieved using a 24 factorial design. The total peptide concentration, the antioxidant activity, and organic and inorganic content were analyzed in the permeate and retentate fraction. The fractions were also tested for their enhanced growth ability and the specific -interferon productivity with CHO cells. Furthermore the retentate and permeate fractions were analyzed by reversed phase-HPLC to characterize the peptide and free amino acid distribution profile. Through the factorial design, the membrane type was shown to have a significant effect on the filtration performance for both yeast extract and Primatone. A significant difference, but similar for both feed sources, was observed for the total peptide transmission with around 10% for the HL membrane and around 30% for the G-10 membrane. The average permeate flux was significantly lower for the G-10 membrane although the G-10 membrane is a loose nanofiltration membrane with a reported 2500 Da MWCO compared to the HL membrane with a reported 300-500 Da MWCO. The total peptide transmission, organic and inorganic content of the fractions for the two feed sources and membrane type were affected differently according to pH and NaCl addition. These results indicate that the two feed sources are of different composition and that nanofiltration is a possible method to fractionate peptides. The bioactivity of the nanofiltration fractions was tested as a nutrient additive to a serum-free media in CHO cells. It was shown that the productivity is not always related to the cell density, as the highest overall specific interferon productivity was achieved for low cell density similar to the hydrolysate free negative control. Furthermore, the retentate fraction of yeast extract separated with the G-10 membrane at a pH of 8 resulted in the highest cell density. According to these results, nanofiltration is a promising method for the enrichment of protein hydrolysates as a supplement for serum in cell culture.

Yellow pigment (YP) concentration is an important quality trait in durum wheat (<i>Triticum turgidum</i> L. var <i>durum</i>) and is comprised primarily of carotenoids. The main objective of our study was to measure the accumulation of carotenoids during the grain fill period to improve our understanding of the physiological basis for differences among durum wheat cultivars. Thirteen cultivars and breeding genotypes with large variation in total YP concentration (<6 µg g-1 to >15 µg g-1) were studied. Spikes were sampled from replicated field plots in 2007 and 2008 near Saskatoon and Swift Current, Saskatchewan, Canada, at 14, 21, 28 and 35 days after heading (DAH). The remainder of each plot was combined at grain maturity for YP and carotenoid analysis. Carotenoids were extracted with 1:1 methanol:dichloromethane (0.1% BHT) and quantified with HPLC. <i>Trans</i> (E)-lutein was the predominant carotenoid at maturity and was detected at 14 DAH in all genotypes. The rate and duration of E-lutein accumulation was variable among genotypes expressing high, intermediate and low YP. The accumulation of all carotenoids was lowest in genotypes expressing low YP, and suggests rate limitations early in the carotenoid biosynthetic pathway. E-zeaxanthin concentrations were highest in mature grain, but no significant differences were detected among genotypes. However, the ratio of E-zeaxanthin to E-lutein was inversely correlated with total YP, suggesting that the â,å branch of lycopene cyclization is favoured over the â,â branch in high-YP genotypes. These results provide insights to the regulation of the carotenoid biosynthetic pathway during grain fill stage in durum wheat and will facilitate breeding for higher carotenoid concentration.

Organic acids, including L- and D-lactate, explain most but not the entire elevated anion gap in diarrhea-associated metabolic acidosis. Also, D-lactate has been implicated in the neurological symptoms associated with this condition. Less-common organic compounds may influence the anion gap and neurological symptoms. This research aimed to characterize and attempt to identify a previously unidentified compound, Compound X, first noted in diarrheic acidotic calves with elevated anion gap (Omole, 1999).<p> High performance liquid chromatography (HPLC) was used to measure Compound X in biological fluids from diarrheic and healthy calves; diarrheic piglets, foals, and human infants; and calves experimentally infused with saline or acid. Attempts were made to identify Compound X using HPLC with tandem and Fourier-transform mass spectrometry.<p> Compound X was significantly higher in diarrheic calf serum (p<0.001) and lower in feces (p<0.001) and rumen fluid (p<0.001) than those fluids from healthy calves. Compound X in serum from acid-infused calves (median peak area ratio = 1.5 1.9) was lower than that of diarrheic calves (median = 4.8) and only slightly greater than that of healthy calves (median = 1.2). Serum Compound X correlated with serum D-lactate in diarrheic and healthy calves combined; however, no such correlation was observed in acid-infused calves. Conversely, a relationship between Compound X and neurological disturbance was present in acid-infused calves, but not in diarrheic calves. In other species, Compound X was highest in diarrheic infants and lowest in diarrheic piglets. Although mass spectrometry and database library searches revealed several compounds as putative matches for Compound X, none of the compounds made sense within the context of acidosis and mammalian biological fluids. Therefore, the identity of Compound X remains unknown.<p> Compound X has been established as a ubiquitous compound(s) present in the biological fluids of mammals. Compound X may be a normal intestinal compound or bacterial metabolite that crosses the intestinal epithelium during diarrhea. In spite of this, Compound X was associated with the neurological manifestations of D-lactic acidosis. Compound X`s identity was not determined, and some reasons for this and future directions are discussed.