Magister Pharmaceuticae - MPharm / Sutherlandia frutescens is a traditional plant medicine widely used in South Africa. Traditionally, the leaves of S. frutescens are mainly used as a tea, but these traditional dosage forms have several disadvantages, including that they are not particularly convenient to prepare and store, encourage dosage inaccuracy and are highly susceptible to microbial contamination. To solve these problems, dried aqueous extract forms, e.g. freeze dried aqueous extract (FDAE) of S. frutescens were prepared, but they, in turn, may still suffer from instability and contain mainly hydrophilic phytoconstituents that are poorly absorbed and delivered for in vivo activity. Modified forms of the FDAE, i.e. the active phytopharmaceutical ingredient (API), may be a better solution. Therefore this study sought to prepare liposomes and phytosomes of the freeze dried aqueous extract of Sutherlandia frutescens, as a means of increasing the total the surface area of the API, thus improving its release and dissolution in gastrointestinal fluids. Liposomes and phytosomes of the FDAE of Sutherlandia frutescens obtained were prepared using a thin film hydration method at ratios of lecithin: S. frutescens (3:1) and phosphatidylcholine: S. frutescens (2:1) respectively. The physical characteristics (i.e. particle size, size distribution, zeta potential, and morphology), of flavonoid glycosides (i.e. sutherlandins A to D; API) as well as content and release profiles of each dosage form (i.e. FDAE liposome or phytosomes) at pH 1.2 and pH 6.8 was determined. A validated HPLC assay was used to determine and compare the flavonoid glycoside content and release profiles of the liposomes and phytosomes. Both liposomes and phytosomes were successfully prepared, in moderate yields (± 30 %, and ± 50 %, respectively), using the thin film hydration method. The liposomes had a significantly smaller size, lower size distribution, higher zeta potential and better stability than the phytosomes (p < 0.05). The phytosomes, however, had significantly higher flavonoid glycoside encapsulation efficiency than the liposomes (±50 % vs ±26 %; p < 0.01). In addition, the release at 120 minutes, of flavonoid glycosides from the liposomes (63%, 58%, 76% and 46% % at pH 1.2, and 78%, 76%, 87% and 89 % at pH 6.8 for sutherlandins A, B, C and D, respectively) was significantly higher and faster than that of the phytosomes (52%, 41%, 51% and 39 % at pH 1.2, and 31% 31%, 33%and 45% % at pH 6.8, for sutherlandins A, B, C and D, respectively). The differences in release were likely due to differences in particle size and size distribution of the two modified API forms. Overall, liposomes and phytosomes can be considered promising vehicles for delayed delivery of herbal crude extracts. Based on its characteristics (i.e. narrower size distribution, and better stability), the liposomes were preferred compared to the phytosomes offering a better kinetic release profile. The phytosomes had higher encapsulation than the liposomes that may be due to complex formation between the API and the lipid.

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

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Hana Pešková Supervisor: PharmDr. Marie Vopršalová, CSc. Title of diploma thesis: Effects of some flavonoids in isolated rat aortic rings Flavonoids with their wide range of effects are known for having positive influence on human health. It mainly concerns significant antioxidant and anti- inflammatory characteristics and cardiovascular diseases incidence reducing effects. The aim of this study was examination of vasorelaxant effects of selected flavonoids from isoflavonoid group: formononetin, isoformononetin, prunetin and ononin, occuring especially in Fabaceae family. The vasorelaxation potential of tested substances was examinated in in vitro conditions on isolated aortic rings of rat. The effect of increasing doses of individual flavonoids on preparation, with intact endothelium after previous precontraction with norepinefrine, was monitored (10-5 M). From measured values, there were DRC curves, EC50 values created and an evaluation was made. According to analysis of the results, the most significat activity has fromononetin (EC50 2,081 . 10-5 M) and isoformononetin (EC50 2,081, 10-5 M). Ononin showed almost no vasodilatory effect.

Aims: Cancer is the first cause of death in the Peruvian population; searching alternative treatments of medicinal plants constitute a promissory field to find new anticancer drugs. The main objective in this study was to evaluate the phytochemical screening, total phenolic content, antioxidant and cytotoxic activity of ethanol extract of Chromolaena laevigata (C. laevigata) on human tumor cell lines. Study Design: The fresh leaves of C. laevigata were soaked with ethanol followed by phytochemical screening using standard methods. Place and Duration of Study: Laboratory of Applied Chemistry, Faculty of Pharmacy and Biochemistry, Universidad Nacional San Luis Gonzaga de Ica, Ica, Peru; Laboratory “Abraham Vaisberg Wolach”, Universidad Peruana Cayetano Heredia, Lima, Peru. Methodology: Phytochemical screening was assessed by using chemical reactives. Total phenolic content (TPC) was developed using Folin Ciocalteu reactive and the antioxidant activity was determined against DPPH and ABTS radicals by spectrophotometry. The cytotoxic activity was determined on human tumor cell lines followed as: MCF-7, H-460, HT-29, M-14, K-562 and DU-145. Results: Phytochemical study confirmed flavonoids and phenolic compounds in ethanol extract. TPC resulted 45.21 ± 3.5 mg of gallic acid equivalent/g of dried extract. The highest antioxidant extract for DPPH and ABTS radical scavenging tests were IC50 = 11.66 ± 1.0 μg/mL, IC50= 12.45 ± 0.50 μg/mL respectively. Ethanolic extracts (μg/mL) showed a low cytotoxicity on human tumor cell lines (CI50 > 20 μg/mL) for DU-145, HT-29, MCF-7 and M-14. Whereas, for H-460, and K562 tumor cell lines showed high cytotoxicity. Conclusion: In our findings, C. laevigata demonstrated a high antioxidant and total phenolic content. The ethanol extract exhibited better cytotoxic effect compared with 5-FU. Hence, This medicinal plant could be effective to prevent chronical diseases as cancer and oxidative stress disorders.

For thousands of years ginger rhizomes are used in traditional Chinese medicine. Because of his significant pungent flavour is commonly used in foods and beverages all over the world. Nowadays many of the beneficial pharmacological positive effects of ginger rhizomes are identified. Their occurrence is due to the high concentration of bioactive compounds. This thesis is focused on analysing of occurrence of bioactive compounds such as polyphenols and flavonoids and their influence on antioxidation and antimicrobial properties of samples of fresh and dried ginger. The results show that for extraction of bioactive compounds in different ginger samples is more suitable to use different types of extraction solvents. The highest antioxidation activity was measured for macerate of 100% bio ginger tea from Sonnentor company in 96% ethanol. Antimicrobial properties were showed only for 100% bio ginger tea against gram-positive bacteria Microccocus luteus.

Flavonoids are natural compounds widely distributed in plant kingdom. They are inseparable from human diet because they showed a protective effect against cancer, stroke and coronary heart diseases related to their antioxidant properties. Therefore, rapid and efficient extraction procedure prior to chromatographic analysis is required. The liquid extraction at elevated temperature and pressure – Pressurized fluid extraction (PFE) and Pressurized hot water extraction (PHWE), present fast, effective and environmentally friendly extraction methods for the determination of flavonoids in plant materials. PFE and PHWE followed by reversed phase high-performance liquid chromatography with UV-visible detection have been utilized for the determination of a group of flavonoids (rutin, myricetin, quercetin, luteolin, apigenin and kaempferol) in the leaves and berries of less common plants. The matrices were extracted by methanol, ethanol and water at higher temperature 40-120 oC and pressure 15 MPa during 15 minutes. The obtained results were compared with conventional Soxhlet extraction and ultrasound-assisted extraction, the same solvents were used. The most effective extraction of selected flavonoids was achieved by PHWE. PFE showed the extraction yields comparable to those of the Soxhlet extraction, and the lowest extraction power was displayed by ultrasound-assisted extraction.

Orientador: Wagner Vilegas / Coorientador: Lourdes Campaner dos Santos / Banca: Isabele Rodrigues Nascimento / Banca: Vera Lucia Garcia Rehder / Banca: João Carlos Palazzo de Mello / Banca: José Arimatéia Dantas Lopes / Resumo: O objetivo desse trabalho foi a investigação da composição química de plantas do Cerrado, aplicando técnicas que permitam uma análise quali e quantitativa rápida e eficaz. As espécies estudadas neste projeto foram Miconia rubiginosa e Miconia stenostachya, pertencentes à família Melastomataceae. As espécies foram coletadas no Estado de São Paulo e os infusos foram preparados e particionados com acetato de etila e água. A fração acetato de etila da M. rubiginosa foi fracionada por Cromatografia em Contracorrente de Alta Velocidade (HSCCC), sendo isolados os compostos (-)-(2R,3R)-epicatequina (Mr1), casuarictina (Mr2), canferol-3-O-β-galactopiranosídeo (Mr3) e schizandrisídeo (Mr4). A fração acetato de etila da M. stenostachya foi fracionada por cromatografia de permeação em gel utilizando Sephadex LH-20, que possibilitou o isolamento da (+)-(2R,3S)-4'-O-metilgalocatequina (Ms1), (-)-(2R,3R)-4'-O-metilepigalocatequina (Ms2), miricetina-3-O-α-rhamnopiranosídeo (Ms3) e (-)-(2R,3R)-epicatequina (Ms4). Foram desenvolvidos métodos de separação dos enantiômeros da naringenina e catequinas por Cromatografia Líquida de Alta Eficiência acoplada a detectores de Dicroísmo Circular e Arranjo de Diodos (HPLC-CD-DAD). Os métodos desenvolvidos forneceram separação adequada com boa resolução. Foram obtidos os espectros de dicroísmo circular (CD) de todas as substâncias isoladas, permitindo a identificação dos enantiômeros. Uma metodologia de clean-up foi desenvolvida para a preparação de amostras para análise por cromatografia quiral. O método que utiliza cartucho de C18 (STRATA Phenomenex®) foi o que apresentou melhor seletividade. O método desenvolvido no HPLC-CD-DAD permitiu identificar a (-)-(2R,3R)-epicatequina no infuso de M. rubiginosa. O mesmo método foi aplicado para investigar a espécie Maytenus obtusifolia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this project was to investigate the chemical composition of Cerrado plants, using techniques that allow a quick and efficient qualitative and quantitative analysis. In this project we studied the species Miconia rubiginosa and Miconia stenostachya which belong to the Melastomataceae family. Miconia species were collected at São Paulo State and water infusions were prepared and partitioned with EtOAc and water. The ethyl acetate fraction of M. rubiginosa was fractioned by High Speed Countercurrent Chromatography (HSCCC), and afforded (-)-(2R,3R)-epicatechin (Mr1), casuarictin (Mr2), kaempferol-3-O-β-galactopyranoside (Mr3), and schizandriside (Mr4). The ethyl acetate fraction of M. stenostachya was submitted to gel permeation chromatography over Sephadex LH-20, that led to the isolation of (+)-(2R,3S)-4'-O-methylgallocatechin (Ms1), (-)-(2R,3R)-4'-O-methylepigallocatechin (Ms2), myricetin-3-O-α-rhamnopyranoside (Ms3), and (-)-(2R,3R)-epicatechin (Ms4). Methods were developed for the enantiomeric separation of naringenin and catechins by High Performance Liquid Chromatography with Circular Dichroism and Diode Array Detectors (HPLC-CD-DAD). The developed methods provided good separation and resolution. CD spectra of all these substances were obtained, allowing the identification of the enantiomers. A clean-up methodology was developed for the preparation of plant samples previous to the chiral HPLC analysis. The method using C18-cartridge (Phenomenex STRATA®) showed the best selectivity. The method developed using HPLC-CD-DAD allowed to identify (-)-(2R,3R)-epicatechin in the infusion of M. rubiginosa. The same method was applied to investigate the commonly used... (Complete abstract click electronic access below) / Doutor

Plant natural products include a diverse array of compounds that play important roles in plant metabolism and physiology. After elucidation of biosynthetic pathways and regulatory factors, it has become possible to metabolically engineer new capabilities in planta as well as successfully engineer whole pathways into microbial systems. Microbial expression systems for producing valuable plant compounds have evolved to incorporate polyculture and co-culture consortiums for carrying out robust biosynthesis strategies. This review focuses on four classes of plant secondary metabolites and the recent advances in generating useful compounds in microbial expression platforms and in plant metabolic engineering. They are the flavonoids, alkaloids, betalains, and glucosinolates.

Maternal immune activation (MIA) can affect fetal brain development and thus behavior of young and adult offspring. Reports have shown that increased Interleukin-6 (IL-6) in the maternal serum plays a key role in altering fetal brain development, and may impair social behaviors in the offspring. Interestingly, these effects could be attenuated by blocking IL-6. The current study investigated the effects of luteolin, a citrus bioflavonoid, and its structural analog, diosmin, on IL-6 induced JAK2/STAT3 (Janus tyrosine kinase-2/signal transducer and activator of transcription-3) phosphorylation and signaling as well as behavioral phenotypes of MIA offspring. Luteolin and diosmin inhibited neuronal JAK2/STAT3 phosphorylation both in vitro and in vivo following IL-6 challenge as well as significantly diminishing behavioral deficits in social interaction. Importantly, our results showed that diosmin (10 mg/kg day) was able to block the STAT3 signal pathway; significantly opposing MIA-induced abnormal behavior and neuropathological abnormalities in MIA/adult offspring. Diosmin's molecular inhibition of JAK2/STAT3 pathway may underlie the attenuation of abnormal social interaction in IL-6/MIA adult offspring.

Eleven subjects completed a clinical trial to determine the safety/tolerability of freeze-dried black raspberries (BRB) and to measure, in plasma and urine, specific anthocyanins-cyanidin-3-glucoside, cyanidin-3-sambubioside, cyanidin-3-rutinoside, and cyanidin-3- xylosylrutinoside, as well as ellagic acid. Subjects were fed 45 g of freeze-dried BRB daily for 7 days. Blood samples were collected predose on days 1 and 7 and at 10 time points postdose. Urine was collected for 12 hours predose on days 1 and 7 and at three 4-hour intervals postdose. Maximum concentrations of anthocyanins and ellagic acid in plasma occurred at 1 to 2 hours, and maximum quantities in urine appeared from 0 to 4 hours. Overall, less than 1% of these compounds were absorbed and excreted in urine. None of the pharmacokinetic parameters changed significantly between days 1 and 7. In conclusion, 45 g of freeze-dried BRB daily are well tolerated and result in quantifiable anthocyanins and ellagic acid in plasma and urine.