Magister Pharmaceuticae - MPharm / Leonotis leonurus, a South African indigenous medicinal plant, is frequently used in the form of a tea. However, this dosage form has many disadvantages. Consequently three L. leonurus solid extract preparations were prepared and explored as possible replacements of the tea form, but very little was known about their physical and chemical stability during storage. The specific objectives were to: (i) prepare a freeze dried aqueous extract (FDAE), 20 % aqueous ethanol (Aq EtOH) extract and calcium alginate beads of the FDAE form of L. leonurus, (ii) characterize the extracts using parameters of select physical and chemical features and, (iii) determine the long-term stability of the extracts. It was hypothesised that the Aq EtOH extract would contain higher levels of chemical marker compounds (marrubiin and leonurine) than the FDAE and calcium alginate FDAE beads of L. leonurus and, that the calcium alginate FDAE beads would have greater stability (i.e. longer shelf-life) than the FDAE and the Aq EtOH extract. The three L. leonurus solid extracts were prepared using accepted published methods. For the physical characterization of the extracts, the organoleptic properties were determined using the natural senses (e.g. sight, smell, taste, etc.) and for chemical characterization, total phenol content (TPC; using the Folin-Ciocalteu reagent method), total flavonoid content (TFC; using aluminium chloride-methanol solution) and antioxidant activity (using the -diphenyl-2-picryl-hydrazyl (DPPH) assay). To establish the long-term stability of the preparations, encapsulated L. leonurus solid extracts was stored in sealed standard plastic containers at four conditions: (A), room temperature of 24 ˚C ± 5 ˚C; (B), fixed temperature of 30˚C ± 5 ˚C and (C), elevated temperature of 40˚C ± 5 ˚C for 6 months, and (D), accelerated stability test conditions of 40˚C ± 5 ˚C / 75 % RH for 4 weeks. Samples of the stored encapsulated preparations were collected periodically and assessed for changes in organoleptic properties, TPC, TFC, antioxidant activity levels and marker compound (i.e. marrubiin and leonurine) levels. The latter was determined by validated HPLC assay. Yields of 19.9, 12.82 and 10.7 % of FDAE, Aq EtOH extract and calcium alginate FDAE beads were obtained, respectively. Physically the calcium alginate beads contained less moisture (1.86 %) than the FDAE (3.77 %) and Aq EtOH (2.91 %). Chemically the FDAE, Aq EtOH extract and calcium alginate FDAE beads respectively had appreciable and similar TPC (i.e.7.86, 7.52 &, 6.94 mg GAE/g; p > 0.05; Anova) and TFC (i.e. 4.30, 4.47 & 3.67 mg QE/g; p > 0.05; Anova) levels, but variable amounts of marrubiin (i.e. 22.5, 17.5, and 0.4 ug/mg plant extract) and leonurine (i.e. 2.0, 1.4 and 0.7 ug/mg plant extract), respectively. The antioxidant activity levels were also different i.e. EC50 values of 7.71, 6.66 and 11.53 mg/mL (student t-test p-value of < 0.0001; ANOVA-test; p< 0.05) for the FDAE, Aq EtOH extract and calcium alginate FDAE beads, respectively. During storage (i.e. stability study) the L. leonurus solid extracts generally remained physically unaffected by temperature (i.e. no significant change in organoleptic features), but when exposed to humidity the FDAE and Aq EtOH extracts showed clear signs of physical degradation i.e. changed from being flaky powders to sticky melted masses, while the calcium alginate beads remained unchanged. Within 1 month storage at RT, 30 °C, 40 °C and 1 week at 40 °C / 75 % RH the TPC of the encapsulated FDAE decreased significantly by 61, 60, 58 and 52 %, respectively, that for the encapsulated Aq EtOH extract by 61, 54, 46 and 50 %, respectively, and for calcium alginate FDAE beads by 66, 71, 59 and 57 %, respectively. Using TPC as a stability parameter all three encapsulated extracts had very short shelf-lives ranging from 1.24 weeks (0.31 months) to 3.72 weeks (0.93 months). Under the same conditions and storage periods (i.e. 1 month & 1 week) the TFC of the encapsulated FDAE decreased significantly by 25, 25, 29 and 66 %, respectively, for encapsulated Aq EtOH extract by 26, 26, 23 and 70 %, respectively, and the calcium alginate FDAE beads by 55, 55, 52 and 64 %, respectively. The results obtained for TFC was thus similar to that obtained for the TPC data. Based on the TFC data all three encapsulated extracts had very short shelf-lives ranging, from 1.56 weeks (0.39 months) to 6.76 weeks (1.69 months). Under the same conditions and storage periods (i.e. 1 month & 1 week) as that used to determine TPC and TFC, the antioxidant activity of the extracts changed little, i.e. decreased by 0.2, 0.1, 0.8 and 2 %, respectively for FDAE, by 0.7 %, 1 %, 0.1 % and 5.3 %, respectively for the Aq EtOH and by 2, 2, 1.4 and 0.8 %, respectively for the calcium alginate FDAE beads. Moreover, based on antioxidant activity, all three encapsulated extracts had relatively long shelf-lives ranging from 15.6 weeks (3.9 months) to 22.4 weeks (5.6 months). Finally, the determination of the stability of the encapsulated L. leonurus extracts stored under stress conditions (i.e. 40 °C / 75 % RH) and based on marker compound levels was unresolved. Between the time of extract preparation and characterisation until start of the stability study the marrubiin levels in the FDAE, Aq. ETOH and calcium beads had decreased from 22.5, 17.5, and 0.4 ug/mg plant extract, respectively, to 0.30, 0.11, 0.30 μg/mg, respectively, and the leonurine levels from 2.0, 1.4 and 0.7 to 0.46, 0.38 and 0.09 μg/mg, respectively and was too low to conduct a meaningful stability study with the developed validated assay. Overall, all three the encapsulated L. leonurus solid extracts studied were clearly very unstable and did not have suitable long-term storage stability. The modification of the freeze-dried aqueous extract of L. leonurus into a calcium alginate bead form seemed to combat physical instability but did not improve the chemical instability of the aqueous extract. It is therefore recommended that the addition of excipients or other post extract modification (e.g. production of phytosomes) be explored to combat the hygroscopicity of L. leonurus FDAE and ultimately improve its overall product stability.

Magister Pharmaceuticae - MPharm / Leonotis leonurus (L. leonurus) and Artemisia afra (A. afra) are two of the most commonly used medicinal plants in South Africa traditionally advocated for use in asthma. However, proper scientific studies to validate these claimed uses are lacking and little is known about the mechanisms for this effect. These plants contain flavonoids, which are reported to have smooth muscle relaxant activity and may be responsible for the activity of these two plants. The objectives of this study were to: (1) determine and compare the flavonoid profiles and levels in A. afra and L. leonurus, (2) compare the respiratory smooth muscle relaxant effects of freeze-dried aqueous extracts of A. afra and L. leonurus and (3) investigate whether K⁺ - channel activation (i.e. KATP channel) is one possible mechanism of action that can explain the effect obtained in traditional use of these two plants. It was hypothesized that: (1) the flavonoid levels and profile of A. afra would be greater than the flavonoid levels and profile of L. leonurus, (2) A. afra would have a more potent respiratory muscle relaxant effect than L. leonurus and (3) A. afra and L. leonurus will inhibit K⁺ - induced contractions in a superior manner than carbachol and histamine - induced contractions. To realize these objectives, freeze-dried aqueous extracts (FDAE) of the dried leaves of the two plants were prepared. A validated HPLC assay was developed and used to identify and determine the levels of luteolin in the plant preparations. Solutions of the plant extracts were studied in the isolated guinea-pig trachea tissue preparation in the presence of carbachol, histamine and KCL. The possible mechanism of action of the two plants was determined by cumulative log dose-response curves (LDRC) for carbachol, histamine and KCL in the absence and presence of 1, 30 and 100 mg/ml solutions of the plant extracts. The flavonoid profile of un-hydrolyzed and hydrolyzed L. leonurus was greater than that of un-hydrolyzed and hydrolyzed A. afra. The levels of free and total luteolin in A. afra FDAE (8.977 ± 0.73 μg/ml and 16.394 ± 0.884 μg/ml, respectively) were significantly (p < 0.001) higher than that in L. leonurus FDAE (0.929 ± 0.066 μg/ml and 3.093 ± 0.531 μg/ml, respectively). L. leonurus and A. afra relaxed tracheal smooth muscles contracted with histamine, KCL and carbachol in a dose dependent manner. The degree of relaxant activity of L. leonurus versus the three inducers of contraction (agonists) could be classified as KCL > carbachol > histamine, with EC₅₀ values of 9.87, 29.34 and 94.76 mg/ml, respectively. The A. afra tracheal smooth muscle relaxant activity was categorized as carbachol > histamine > KCL, with EC₅₀ values of 13.93, 15.47 and 19.88 mg/ml, respectively. Overall, A. afra which contained the higher levels of luteolin, was more potent at relaxing the guinea pig tracheal smooth muscle than L. leonurus. Collectively, the results confirm that aqueous solutions of A. afra and L. leonurus as used in local traditional practice have potent but different degrees of bronchodilator activities that could be useful in the treatment of asthma, and that these actions may be related to each plant's luteolin (or flavonoid) levels. Moreover it is very unlikely that KATP channels are primarily responsible for the actions of A. afra and L. leonurus, but rather that more than one mechanism of action is involved in the tracheal smooth muscle relaxant effects of these two plants. / National Research Foundation

The total water uptake, rate of water uptake, extract release volume and maximum shear force were measured on a series of samples of irradiated freeze-dried beef. Forty seven pieces of round steak (2.5 cm x 2.5 cm x 10.4 cm or 1" x 1" x 4") were irradiated at one, three and five megarad. The control samples were not irradiated. Half of the samples were irradiated when fresh, and the other half were irradiated after freeze drying. This procedure has been defined as the "fresh-dry" irradiation sequence throughout the report. The samples were frozen in an air blast at two temperatures (-22.2°C and -56.1°C). Freeze-drying was carried out below 300 microns of Hg and a maximum shelf temperature of 15.6°C (60°F). There appears to be three phases of water uptake: 1) A very rapid, almost instantaneous, absorption. 2) A more gradual uptake (called Part.1 in the report). 3) A relatively slow asymptotic approach to an equilibrium condition (Part 2). These two last phases are shown to be straight lines when the logarithm of the water uptake is plotted against the logarithm of the immersion time. Irradiation level has no significant effect on the final water content or on the slow asymptotic absorption (Part 2) or the extract release volume, but has a significant effect on the gradual water uptake (Part 1) and on the shear press force. Fresh-dry irradiation sequence (and freezing rate) have a significant effect on the total water uptake and on the slow asymptotic water (Part 2) uptake, but not on the gradual water uptake (Part 1), or on the extract release volume or on the shear press forces. Freezing rates have a significant effect on the total water uptake, but not on the slow asymptotic water uptake (Part 2), on the gradual water uptake (Part 1), on the extract release volume or on the shear press forces. The highest total water uptake was found for the meat irradiated when fresh, and slow frozen at -2 2.2°C. The mechanism of the gradual absorption appears to follow a phenomena of water flow, as evidenced by the straight line relationship found in the plots of logarithm water uptake versus logarithm immersion time. / Land and Food Systems, Faculty of / Graduate

A total of seven anthocyanin pigments were observed in both paper and thin layer chromatograms of the fresh and freeze-dried compressed samples stored for O month whereas the freeze-dried compressed samples stored at 70 F and 100 F for 6 months showed the retention of three to six pigments. All of those seven pigments were unstable and cyanidin-3- (2G- xylosylrutinoside) was the least stable pigment. The separation of anthocyanina by disc gel electrophoresis was studied for the first time. Disc electropherograms of fresh and freeze-dried com-pressed sour cherries stored at O month revealed the presence of eight bands whereas the freeze-dried compressed sour cherries stored at 70 F and 100 F for 6 months showed the retention of three to four bands. In further study, this technique could be helpful for the separation of anthocyanins in other fruits. The study indicated that the fresh samples had anthocyanin content higher than those of the freeze-dried compressed samples stored at 70 F and 100 F for 6 months and also showed that the degradation of anthocyanins is greater at the higher storage temperature with longer storage periods.

Thesis. 1975. M.S.--Massachusetts Institute of Technology. Dept. of Nutrition and Food Science. / Bibliography: leaves 109-112. / M.S.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Nutrition and Food Science, 1979. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 153-157. / Ph.D.

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Tendo em vista os vários problemas envolvendo a metodologia correntemente utilizada para a determinação da viabilidade no controle de qualidade da vacina BCG liofilizada, foi levado a efeito um estudo a respeito do assunto. Foi procedido um estudo de métodos alternativos a serem empregados em procedimentos de controle de qualidade da vacina BCG liofilizada. Com esse objetivo foi desenhado e construído um polarógrafo de eletrodo de oxigênio constituído de eletrodo de platina e o de referência de Ag-AgCl embutido em peça única e ainda dotado de sensor de temperatura, com circuito eletrônico permitindo a medida de consumo de oxigênio. Levou-se a efeito, então, estudo comparativo de viabilidade de vacina BCG liofilizada pela medida polarográfica de consumo de oxigênio em comparação à manométrica pelo clássico método de Warburg e o método de contagem de colônias (UFC). A análise de 66 diferentes lotes de vacina BCG liofilizada produzidas pelo Instituto Butantan, São Paulo, e pela Fundação Ataulpho de Paiva, Rio de Janeiro, demonstrou que a técnica polarográfica revelou ser altamente reprodutível e muito mais conveniente que os métodos manométrico e de contagem de colônias. Verificou-se que entre o método polarográfico e o manométrico e UFC os resultados da regressão linear mostraram correlação positiva de alta intensidade e significante ao nível de a= 0,05, permitindo indicar o método polarográfico descrito, como procedimento de escolha para operações de controle de qualidade em laboratórios de produção de vacina BCG bem como em Unidades de Saúde, para determinações adequadas de viabilidade de lotes de vacinas, a serem utilizadas para fins de imunização. / Considering the several problems regarding the procedures currently employed in the assays o f the control of quality, and the viability of the freeze-dried BCG vaccines, a study on various methods used for this purpose was carried out. Aiming to introduce a much more suitable, sensitive and less time consuming method than the classic Warburg technique and the colony count test (CFU) bacteriological procedure, currently used, a new oxygen electrode polarograph was designed with the aim of employing it for the study of the viability of the freeze-dried BCG vaccine. This polarograph electrode was built in a single piece containing the platinun and theAgAgCl reference electrode besides a temperature sensor. By using this assembly a comparative study was carried on the viability of the freeze-dried BCG vaccine assayed by the polarographic method in comparison with the manometric and the CFU methods. The analysis of the viability of 66 different batches of freeze-dried BCG vaccines produced at the Butantan Institute, São Paulo, and at the Ataulpho de Paiva Foundation, Rio de Janeiro, demonstrated that the polarograpbic technique revealed to be highly reliable and much more convenient than both the manometric and the CFU methods: it has been found out that between the polarographic and the manometric and the CFU, the linear regression sbowed a high intensity positive correlation and significant at the level of a = 0,05. In this way, the polarographic method could be considered as a choice method for the control of quality procedure as well as for the establishment of the viability of the batches of vaccines to be used for human immunization in Units of the Health Department.

Tendo em vista os vários problemas envolvendo a metodologia correntemente utilizada para a determinação da viabilidade no controle de qualidade da vacina BCG liofilizada, foi levado a efeito um estudo a respeito do assunto. Foi procedido um estudo de métodos alternativos a serem empregados em procedimentos de controle de qualidade da vacina BCG liofilizada. Com esse objetivo foi desenhado e construído um polarógrafo de eletrodo de oxigênio constituído de eletrodo de platina e o de referência de Ag-AgCl embutido em peça única e ainda dotado de sensor de temperatura, com circuito eletrônico permitindo a medida de consumo de oxigênio. Levou-se a efeito, então, estudo comparativo de viabilidade de vacina BCG liofilizada pela medida polarográfica de consumo de oxigênio em comparação à manométrica pelo clássico método de Warburg e o método de contagem de colônias (UFC). A análise de 66 diferentes lotes de vacina BCG liofilizada produzidas pelo Instituto Butantan, São Paulo, e pela Fundação Ataulpho de Paiva, Rio de Janeiro, demonstrou que a técnica polarográfica revelou ser altamente reprodutível e muito mais conveniente que os métodos manométrico e de contagem de colônias. Verificou-se que entre o método polarográfico e o manométrico e UFC os resultados da regressão linear mostraram correlação positiva de alta intensidade e significante ao nível de a= 0,05, permitindo indicar o método polarográfico descrito, como procedimento de escolha para operações de controle de qualidade em laboratórios de produção de vacina BCG bem como em Unidades de Saúde, para determinações adequadas de viabilidade de lotes de vacinas, a serem utilizadas para fins de imunização. / Considering the several problems regarding the procedures currently employed in the assays o f the control of quality, and the viability of the freeze-dried BCG vaccines, a study on various methods used for this purpose was carried out. Aiming to introduce a much more suitable, sensitive and less time consuming method than the classic Warburg technique and the colony count test (CFU) bacteriological procedure, currently used, a new oxygen electrode polarograph was designed with the aim of employing it for the study of the viability of the freeze-dried BCG vaccine. This polarograph electrode was built in a single piece containing the platinun and theAgAgCl reference electrode besides a temperature sensor. By using this assembly a comparative study was carried on the viability of the freeze-dried BCG vaccine assayed by the polarographic method in comparison with the manometric and the CFU methods. The analysis of the viability of 66 different batches of freeze-dried BCG vaccines produced at the Butantan Institute, São Paulo, and at the Ataulpho de Paiva Foundation, Rio de Janeiro, demonstrated that the polarograpbic technique revealed to be highly reliable and much more convenient than both the manometric and the CFU methods: it has been found out that between the polarographic and the manometric and the CFU, the linear regression sbowed a high intensity positive correlation and significant at the level of a = 0,05. In this way, the polarographic method could be considered as a choice method for the control of quality procedure as well as for the establishment of the viability of the batches of vaccines to be used for human immunization in Units of the Health Department.

The açaí berry has grown in popularity for dieters and the health conscious. The berry contains high levels of antioxidants. The main anthocyanins are cyanidin-3-rutinoside and cyanidin-3-glucoside. The berries also contain vitamins and nutrients that help boost energy and alertness, and fatty acids that help maintain normal cholesterol levels. As a result of the health benefits of the constituents, the berries are chosen for obtaining the active ingredients by extraction. The solids left over after extraction still contain nutrients and useful components. There is a possibility of fermenting this residual and creating an açaí berry wine. Açaí berry wine is another new product on the market. The extracts and the wine are analyzed using Fourier transform infrared spectroscopy (FTIR). In an effort to utilize every part of the berry, the residual from the extract is also successfully fermented. Pilot plant studies are conducted utilizing supercritical carbon dioxide, an ethanol entrainer to increase its solubility, and subcritical water to extract components from freeze dried açaí berry. There is much potential and flexibility in the process, which effectively extracted lipids from the berry leaving behind anthocyanins without solvent residue.