O arroz integral pigmentado (vermelho ou preto) (Oryza sativa, L.) é saudável. Ambos são nutritivos, possuem elevado teor de compostos bioativos, e apresentam características sensoriais diferenciadas ao arroz integral branco. O grande destaque sob o ponto de vista de composição química é o seu elevado teor de compostos fenólicos, associado à alta capacidade antioxidante. A demanda por arroz pigmentado ainda é reduzida, porém, existe uma tendência e o interesse em agregar valor a esses grãos, produzindo alimentos alternativos como flocos de arroz, cereais matinais e snacks. O objetivo deste estudo foi desenvolver produtos extrusados expandidos a partir de duas novas cultivares comerciais de arroz integral preto (SCS 120 ÔNIX) e vermelho (SCS 119 RUBI), provenientes da Epagri/SC. Na otimização do processo considerou-se as melhores propriedades tecnológicas e a retenção de compostos bioativos. Foi investigado o efeito do processo de extrusão nos fitoquímicos e sua atividade antioxidante, antes e após a extrusão. Além disso, a aceitabilidade de dois produtos finais foi realizada com 150 consumidores. O arroz preto apresentou teores significativamente maiores de macronutrientes (proteínas, fibras e minerais) do que o arroz vermelho. O teor de compostos fenólicos totais (CFT) e flavonoides no arroz preto foi de 25% (569,3 mg eq. ác. ferúlico/100g) e 44% (496,9 mg eq. catequina/100 g), respectivamente, superior ao do arroz vermelho. Os flavonoides são representados principalmente por antocianinas no arroz preto e por proantocianidinas no arroz vermelho. O arroz preto apresentou maior atividade antioxidante. A partir do delineamento composto central rotacional as condições ótimas de extrusão foram de 15,5 e 16% de umidade e de 159 e 150ºC de temperatura, para o arroz preto e vermelho, respectivamente. O processo de extrusão resultou em perdas drásticas de TPC, flavonoides, proantocianidinas e o conteúdo remanescente não foi maior que 14% no arroz vermelho e a atividade antioxidante residual também foi baixa. O arroz preto também foi afetado significativamente pela extrusão mas em menores proporções. A concentração após a extrusão foi de 32 % (TPC), 22% (flavonoides), 11% (antocianinas) e a atividade antioxidante remanescente de 40% e 30% pelos métodos de ORAC e DPPH, respectivamente. Quanto à análise sensorial, os extrusados obtidos de arroz preto e vermelho não tiveram diferença significativa na aceitação (p≤0,05). As notas médias atribuídas por 150 provadores para todos os atributos avaliados ficaram situadas no intervalo de 4,8 a 7,7 (correspondente as categorias \"nem gostei/nem desgostei\" a \"gostei muito\") da escala hedônica de 9 pontos. Altos valores de aceitabilidade (maiores que 70%) foram observados para formato, tamanho, cor e crocância no cereal seco e consistência no cereal com leite. A menor aceitabilidade (60%) foi notada no sabor. Isto pode ser explicado pela ausência de aromatizante e/ou edulcorante na formulação. Ainda, foi observado que 26% dos provadores certamente comprariam ambos os extrusados. A farinha de arroz pigmentado é uma alternativa para a elaboração de cereais matinais extrusados na qual apresentam coloração própria, boas características nutricionais e sensoriais, além de ser um produto isento de glúten. / Whole pigmented rice (black and red) (Oryza sativa, L.) is a healthy food. Both types present are nutritious and present high contents of bioactive components, and show distinguishing sensorial properties, unlike those found in whole white rice. However, the most noticeable difference regarding the chemical composition of black and red rice is their high content of phenolic compounds, associated to a high antioxidant activity. Consumption of pigmented rice is still low, but there is an increasing trend and interest in adding value to pigmented grains, producing alternative food products such as rice flakes, breakfast cereals and snacks. Then, the objective of this study was to produce extruded rice from whole grain of black (SCS 120 ÔNIX) and red rice (SCS 119 RUBI). Rice cultivars were grown at experimental fields of Epagri/SC. The conditions of the extrusion process were optimized to achieve the best technological properties and to attain the retention of bioactivties. The phytochemical composition antioxidant activity and technological properties of the pigmented rice flour were investigated before and after extrusion. In addition, the acceptability of the two selected final products was assessed by 150 consumers. The black rice showed significantly higher contents of macronutrients (protein, fiber and minerals) than red rice. The total phenolic compounds (TPC) and total flavonoids in black rice were also 25% (569.3 mg eq. ac. ferulic/100 g) and 44%, (496.9 mg eq. catechin/100 g) respectively, higher than in red rice. In black rice flavonoids were represented mainly by anthocyanins, while in red rice the main flavonoids were proanthocyanidins. Black rice showed the highest antioxidant activity. The predicted optimum extrusion conditions were settled down at 15.5 and 16 % of moisture and 159 and 150ºC of temperature for black and red rice, respectively. The extrusion process induced a drastic loss of TPC, flavonoids and proanthocyanidins and remaining contents were not higher than 14% in red rice and residual antioxidant activity was also very low. Black rice was also significantly affected by the extrusionm but in a lesser degree. The concentration after extrusion was 32% (TPC), 22 % (flavonoids), 11% (anthocyanins) and a remaining 40 % and 30 % antioxidant activity by ORAC and DPPH methods, respectively. Consumer acceptability of extrudates was similar and the average scores awarded were located between 4.8 to 7.7 (corresponding to the categories \"not liked/disliked to \"liked so much\") for all attributes evaluated in the 9-point hedonic scale. High acceptability scores (higher than 70 %) were observed for shape, size, color and texture hardness in dry extrudates and consistency in extrudates with milk. The lowest acceptability (60 %) was noted for flavor. It could be explained because of the extrudates were not flavoured. Furthermore, it was shown that 26 % of consumers certainly would buy both extrudates. The pigmented rice flour demonstrated to be a good alternative to produce extruded breakfast cereals which had his own color, showed good nutritional and sensory properties, besides being a gluten-free product.

Os cálices do hibisco possuem uma grande quantidade de compostos bioativos responsáveis pela sua atividade antioxidante. O presente trabalho teve como objetivos a obtenção de extratos contendo esses compostos bioativos em solução aquosa com 2 % de ácido cítrico por extração assistida por micro-ondas (EAM) e o encapsulamento desses extratos por atomização e liofilização utilizando polidextrose (PD), proteína isolada do soro de leite (WPI) e a mistura destes na concentração de 10 %. Previamente foram estudados três métodos de extração, o primeiro usando somente EAM a 200, 300 e 700 W de potência, e tempos de 2, 5 e 8 minutos; o segundo consistiu de dois períodos, a extração aquosa ácida com tempos de 1, 2, 4, 6, 18 e 24 horas seguida de EAM nas potências de 200, 300 e 700 W; o terceiro consistiu de EAM seguida de extração aquosa ácida, nos mesmos tempos e potências citados para o segundo método. Os resultados indicaram que a melhor condição do primeiro método foi a 700 W e 8 min, do segundo método realizada a 6 horas de extração aquosa ácida, seguida de EAM a 700 W por 8 min e do terceiro método, EAM a 700 W por 8 min seguida de 6 horas de extração aquosa ácida. Quando os três métodos foram comparados, a melhor condição de extração foi aquela obtida no terceiro método: 1,63 mg delfinidina-3-sambubiosídeo · g-1; 29,62 mg EAG · g-1; 133,25 μmol ET · g-1 para antocianinas, fenólicos totais e atividade antioxidante por ABTS, respectivamente. Para avaliar o efeito da extração obtida somente por EAM, os extratos obtidos por extração exaustiva com metanol por 25 min e EAM a 700 W e 8 min foram quantificados por HPLC-DAD-ESI-MS/MS, sendo encontrados 13 compostos (6 ácidos fenólicos, 2 antocianinas e 5 flavonóides derivados da quercetina, kaempferol e miricetina). Os compostos fenólicos majoritários foram o ácido 3-cafeoilquínico (2,58 e 1,32 mg · g-1) e ácido 5-cafeoilquínico (1,71 e 0,90 mg · g-1) para extração exaustiva e EAM, respectivamente. Esse mesmo extrato (700 W e 8 min) foi encapsulado por atomização (160 °C) e liofilização (- 68 °C por 54 horas). Os pós obtidos foram avaliados quanto aos teores de compostos fenólicos totais, antocianinas monoméricas totais, atividade antioxidante (ABTS, DPPH e HRSA), medidas por análises espectrofotométricas, atividade de água, umidade, higroscopicidade, solubilidade, eficiência de encapsulação, cor, análise termogravimétrica, temperatura de transição vítrea, espectroscopia de infravermelho com transformada de Fourier (FTIR) e microestrutura (MEV). Os pós atomizados tiveram menor atividade de água (0,14 a 0,17), umidade (3,4 a 4,5 %), higroscopicidade (23,9 a 34,1 %), solubilidade (86 a 98,2 %) e eficiência de encapsulação (51,62 a 84,52 %) do que os pós liofilizados. Os resultados do FTIR mostraram que os encapsulantes não interagiram quimicamente, visto que não foram observados mudanças na frequência dos picos; as provas termogravimétricas indicaram que os pós apresentaram a mesma tendência nas perdas de massa. Na análise de microestrutura foi observado um melhor desempenho nas micropartículas atomizadas com PD, as quais mostraram partículas mais esféricas e sem tendência de atração e aderência entre si. Foram obtidas retenções de 38 a 77 % para antocianinas monoméricas totais, 42 a 89 % para compostos fenólicos totais, e entre 33 e 90 % para atividade antioxidante nos pós obtidos. O pó encapsulado liofilizado com 10 % de polidextrose mostrou uma maior retenção de antocianinas (77 %), atividade antioxidante por DDPH (90 %) e HRSA (74 %), entretanto com maior higroscopicidade (39,4 %). As provas aceleradas de estocagem (umidades relativas de 75 e 90 % em temperaturas de 40 e 60 °C) realizadas em todos os pós encapsulados, após 30 dias, indicaram que o tratamento liofilizado com 10 % de PD foi o que apresentou melhores resultados a essas condições, retendo 75 % dos compostos fenólicos, com atividades antioxidantes medidas por ABTS, DPPH e HRSA de 75, 90 e 74 %, respectivamente, existentes no extrato original. O pó obtido tem potencial para aplicação em alimentos, portanto, devido ao hibisco ser uma matriz com ampla composição de compostos bioativos. / The hibiscus calyces contend a high quantity of bioactive compounds responsible for their antioxidant activity. The present paper was aimed the production of extracts containing those bioactive compounds in acidified aqueous solution 2 % of citric acid by microwave assisted extraction (MAE) and the encapsulation of those extracts by spray drying and freeze-drying using polydextrose (PD), whey protein isolate (WPI) and their mixture in the concentration of 10 %. Previously three methods of extraction were studied, the first using only MAE at 200, 300 and 700 W of power, and times of 2, 5 and 8 minutes; the second consisted of two steps, the acid aqueous extraction with times of 1, 2, 4, 6, 18 and 24 hours followed by MAE at the powers of 200, 300 and 700 Watts; the third consisted of MAE followed by acid aqueous extraction, in the same times and powers mentioned for the second method. The results indicated that the best condition of the first method was 700 W and 8 minutes, the second method performed at 6 hours of acid aqueous extraction, followed by MAE at 700 W for 8 minutes and the third method, MAE at 700 W for 8 minutes followed by 6 hours of acid aqueous extraction. When the three methods are compared, the best condition of extraction was obtained in the third method: 1.63 mg delphinidin-3-sambubioside · g-1; 29.62 mg GAE · g-1; 133.25 μmol TE · g-1 for total monomeric anthocyanins, total phenolic compounds and antioxidant activity by ABTS, respectively. To evaluate the effect of the extraction obtained only by MAE, the extracts obtained by exhaustive extraction with methanol for 25 minutes and MAE at 700 W and 8 minutes were quantified by HPLC-DAD-ESI-MS/MS, was found 13 compounds (6 phenolic acids, 2 anthocyanins and 5 flavonoids derived from quercetin, kaempferol and myricetin). The phenolic compounds majorities were acid 3-caffeoylquinic (2.58 e 1.32 mg · g-1) and acid 5-caffeoylquinic (1.71 e 0.90 mg · g-1) for exhaustive extraction and MAE, respectively. That same extract (700 W and 8 minutes) was encapsulated by spray drying (160 ºC) and freeze-drying (- 68 °C for 54 hours). The obtained powders were evaluated about the levels of total phenolic compounds, total monomeric anthocyanins, antioxidant activity (ABTS, DPPH e HRSA), measured by spectrophotometric analysis, water activity, moisture, hygroscopicity, solubility, encapsulation efficiency, color, thermogravimetric analysis, glass transition temperature, Fourier transform infrared spectroscopy (FTIR) and microstructure (MEV). The spray dried powders had lower water activity (0.14 to 0.17), moisture (3.4 to 4.5 %), hygroscopicity (23.9 to 34.1 %), solubility (86 to 98.2 %) and encapsulation efficiency (51.62 to 84.52 %) than the freeze-dried powders. The results of FTIR showed that the encapsulants did not interact chemically, since changes were not observed on the frequency of the peaks; the thermogravimetric tests indicated that the powders presented the same tendency on the mass loss. On the microstructure analysis a better perform was observed on the spray dried microparticles with PD, which showed more spherical particles and with no tendency of attraction and adherence between them. Were obtained retentions of 38 to 77 % for total monomeric anthocyanins, 42 to 89 % for total phenolic compounds and between 33 and 90 % for antioxidant activity in the obtained powders. The encapsulated power by freeze-drying, with 10 % of polydextrose, was showed higher retention of anthocyanins (77 %), antioxidant activity by DDPH (90 %) and HRSA (74 %), however with higher hygroscopicity (39.4 %). The accelerated tests of storage (relative humidity of 75 and 90 % in temperatures of 40 and 60 ºC) performed in all the encapsulated powders, after 30 days, indicated that the freeze-drying treatment with 10 % of PD has the best behavior in those conditions, retaining 75 % of the phenolic compounds, with antioxidant activities measured by ABTS, DPPH and HRSA of 75, 90 and 74 %, respectively, present in the original extract. The obtained powder has potential for application in foods, therefore, due to the hibiscus being a matrix with ample composition of bioactive compounds.

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2015 / Antioxidants play an important role in living organisms to control level of free radicals and other reactive molecules in the body to reduce oxidative damage. Synthetic antioxidant compounds are used in food industries as food additives to boost our immune systems. These compounds are associated with a number of critical side effects including liver damage and carcinogenesis. Scientists are also concerned about microorganisms that have developed resistant genes against current antibiotics used in hospitals. The aim of the study was to isolate and characterize bioactive compounds from Ricinus communis leaves with activity against Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). Consequently, medicinal plants are studied and considered for their efficacy and safety, because they possess bioactive compounds with various biological activities. Leaves of R. communis were collected at the University of Limpopo, Turfloop campus in Limpopo province, South Africa. The leaves were dried and milled to a fine powder. A number of trial extraction methods were employed using various solvents of different polarities on a fine powder leaves to identify the best extraction method. Plant extracts were analyzed by thin layer chromatography (TLC) developed in four mobile phases. To detect separated phytochemical compounds, TLC plates were sprayed with vanillin- sulphuric acid in methanol and heated at 110oC for optimal colour development. Qualitative antioxidant activity was determined by using 2, 2–diphenyl-1-picrylhydrazyl (DPPH) assay on TLC plates. Quantitative antioxidant activity was determined by measuring percentages scavenging activity of DPPH and 2, 2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radical molecules by plant extracts. Antibacterial activity of all extracts was quantified by a serial microbroth dilution method while bioautography was used in qualitative analysis of the active compounds. Cytotoxicity effect of R. communis extracts was evaluated using tetrazolium-based calorimetric assay on human Caucasian skin fibroblast (Bud-8) cell line. Anti-inflammatory activity was assessed using phagoburst kit on Raw 264.7 macrophages cell line. Pure compounds were subjected to nuclear magnetic resonance spectroscopy for 1H, 13C and DEPT experiments to elucidate structures of compounds. 2 During extraction process, methanol was the best extractant, extracting greater amount of extracts than any of the other solvents. Serial exhaustive extraction method was selected as the best extraction method for extracting compounds from ground plant materials. In quantitative antioxidant assays, chloroform and methanol extracts had highest percentage scavenging activity against DPPH free radicals compared to other extracts and vitamin C. Methanol extract had the highest percentage scavenging activity of ABTS free radicals and minimum percentage scavenging activity was in hexane extract. Acetone, ethyl acetate and ethanol extracts showed strong antioxidant activity against DPPH free radicals in qualitative antioxidant assay on TLC plates. In quantitative antibacterial assay, crude extracts showed lowest minimum inhibitory concentration value of 0.13 mg/ml against all tested organisms and the highest was 1.05 mg/ml. Hexane extracts revealed potent antibacterial activity against all tested microorganisms on bioautograms. Hexane and acetone extracts also revealed anti-inflammatory activity and have ability to reduce oxidative stress. In cytotoxicity effect of plant extracts, Methanol extracts had lethal concentration for 50% of the cells (Lc50) of 784 μg/ml on Human Caucasian skin fibroblast (Bud-8) cell line while hexane extracts had Lc50 of 629 μg/ml. Plant extracts with high Lc50 are low toxic to normal cell line and preferable to work with for drug development. Bioassay-guided fractionations results in successful isolation of three antioxidant and two antibacterial compounds from R. communis using column chromatography. Isolated compounds were tested for their biological activities using qualitative DPPH assay on TLC plates for antioxidant activity and bioautography for antibacterial activity. Antioxidant compounds showed strong antioxidant activity after spraying with DPPH in methanol and antibacterial compounds showed less activity compared to the crude extracts. The study suggests the use of crude extracts to fight against pathogenic microorganisms compared to pure compounds. Compound 4 was successful identified as the mixture of stigmasterol and β-sitosterol. The present study recommends the use of R. communis leaves as the potential source of antioxidant, antibacterial and anti-inflammatory compounds. The study serves as a scientific proof for use of this plant in traditional medicine for treatment of various ailments.

The presented diploma thesis is focused on the study of composition and biological effects of some super-foods. Theoretical part deals with basic information about chosen superfoods and their bioactive substances. Further, theoretical part describes the overview of vesicular systems used for encapsulation and the most common methods of particle characterization. A brief review of cell cultures and cultivation of human cells is presented as well as methods for cytotoxicity a genotoxicity testing. In the experimental section, aqueous and ethanol extracts of super-foods were prepared. These extracts were then encapsulated into liposomal and combined PHB particles. Super-food extracts were characterized by spectrophotometrical methods in order to determine the content of polyphenols, flavonoids, anthocyanins, carotenes, chlorophyll, tannins, and antioxidant activity. The physico-chemical characteristics of prepared liposomal and combined particles were determined too. The particles with encapsulated extracts were further tested using the MTT assay and SOS chromotest to describe their potential cytotoxic and genotoxic effects.

Production and consumption of edible berries are increasing rapidly in the United States, mostly due to their superior flavor profile, and popular diet-related value with their human health relevant bioactives and nutritional benefits. However, bioactive and nutritional qualities, especially human health protective phenolic antioxidants and associated non-communicable chronic disease (NCD) relevant health benefits of berries vary widely among accessions/cultivars and due to different production practices (organic vs conventional). Therefore, the aim of this thesis was to screen and select high phenolic and high antioxidant serviceberry and blackberry accessions/cultivars and to investigate the effect of different weed management and fertilization (organic vs. conventional) practices on phenolic bioactive linked antioxidant and anti-diabetic properties of blackberry using in vitro assay models. Overall, high phenolic-bioactive linked antioxidant and anti-hyperglycemic properties were observed in both serviceberry and blackberry accessions/cultivars and further for blackberry it was significantly higher under organic weed management and fertilization practices.

Global food and nutritional insecurities, public health challenges of diet-linked non-communicable chronic diseases (NCDs), and rapid climate change-linked agricultural production challenges are interconnected and require urgent attention. Therefore, to address these complex and interconnected challenges, it is essential to advance robust and resilient strategies based on sustainable agricultural production practices, wider integration of nutritionally-balanced plant-based foods in the diet, improvement of human health-targeted nutritional qualities, post-harvest preservation qualities and food processing optimization. Therefore, food plants that are climate resilient and rich source of human health protective nutritional bioactives, such as sweet potato are ideal dietary targets for advancing global food and nutritional security solutions, while also addressing emerging NCD-linked health challenges. Sweet potatoes are rich source of stress protective phenolic bioactives with dual functional benefits relevant for resilience to climate change and countering diet-linked NCD challenges. However, the phenolic bioactive compounds and associated health protective functionalities of sweet potatoes vary widely between different flesh color and cultivars, due to different pre-harvest production practices, post-harvest storage conditions, and with different food processing strategies. Therefore, the aim of this dissertation was to screen sweet potato cultivars of different flesh color (off-white, orange, purple) and optimizing different food processing strategies based on optimum phenolic bioactive-linked antioxidant, anti-diabetic and anti-hypertensive properties using metabolically-targeted in vitro assay models. Overall, high soluble phenolic-linked antioxidant activity was observed in purple-fleshed cultivar, while high type 2 diabetes relevant anti-hyperglycemic and anti-hypertensive properties were observed in orange and white-fleshed sweet potatoes. Additionally, improvement in stability and retention of phenolic bioactives and associated functionalities were present in bio-transformed sweet potatoes after fermentation with beneficial lactic acid bacteria (LAB). Furthermore, food processing (deep-frying, baking, steaming, and boiling) optimization studies revealed optimum food processing conditions (cooking temperature, cooking time, and sweet potato sample size) based on higher retention of phenolics and associated antioxidant and anti-hyperglycemic functionalities. We also advanced metabolically-driven elicitation strategy based on the conceptual foundation of dual functional benefits of phenolic compounds to improve wound-healing in bruised potato tubers through stimulation of redox-linked pathway (pentose phosphate pathway) regulation associated with stress-protective phenolic biosynthesis and antioxidant enzyme responses.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12822号 / 論農博第2795号 / 新制||農||1025(附属図書館) / 学位論文||H26||N4817(農学部図書室) / 31309 / 京都大学農学研究科食品生物科学専攻 / (主査)教授 喜多 恵子, 教授 三上 文三, 教授 栗原 達夫 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p>Natural products, along with natural product derivatives, are known to be at the root of the development of many pharmaceuticals, oftentimes showing unique bioactivity against interesting targets. Specifically, natural products containing furans show activity against a variety of diseases including fungal infections, and cancers. It is hypothesized that unknown natural products containing furans could show more potent or other biological activities. However, it is challenging to discover and isolate these small molecules from cell supernatant. The work described herein showcases the development of a molecular probe that can covalently attach to furan moieties via a [4 + 2] Diels-Alder cycloaddition, making them easily identifiable on liquid chromatography mass spectroscopy (LC-MS). The molecular probe, which undergoes this reaction with a variety of furans, was designed with both a UV-tag and a mass tag to enable easy identification. The probe has been tested with a variety of purified furans, including natural products, methylenomycin furan (MMF) hormones, and MMF derivatives. Moreover, work has begun to test the molecular probe in cell supernatants. </p>

Bioactive compounds, such as curcumin, lutein, coenzyme Q10, β-carotene, cholecalciferol, astaxanthin, and β-sitosterol, have antioxidant and anti-inflammatory properties that promote health, but their low solubility, fast metabolism, and degradation have made it difficult to fully harness their potential. Encapsulation techniques, such as nano and microencapsulation using food-based biopolymers, have been employed to address these challenges. However, research efforts in protein-based delivery have mainly focused on encapsulation without considering structural, physicochemical, and matrix compatibility, which is tedious, unsustainable, and not cost-effective. Hence, this thesis reports the structural basis of guest-host interaction in the gastrointestinal delivery of lipophilic bioactive compounds using protein-based vehicles. This research employed fluorescence quenching techniques to estimate the influence of protein modification, fractionation and ionic strength on the nature and strength of interactions between protein and bioactive compounds. Morphological examination was carried out with transmission electron microscopy, confocal and widefield fluorescence microscopy whereas the sizes of the nano and micro-complexes was measured with dynamic light scattering techniques. Thermal stability was measured with differential scanning calorimetry and functional group characterization done with Fourier Transform infrared spectroscopy. Encapsulation efficiency was estimated by UV-Visible spectroscopy whereas in vitro bioactive compound release study was carried out in simulated salivary, gastric and intestinal fluids. Cytotoxicity assessment was estimated by calcein leakage assay. The study showed that protein modification affects the strength of protein-curcumin interaction and encapsulation efficiency. Pea protein succinylation increased electrostatic interaction with chitosan but decreased protein-curcumin interaction. Pea glutelin, albumin and globulin fractions showed different binding strengths with curcumin and the protein hydrophobicity and encapsulation efficiency correlated positively with the binding strength. The study also investigated the impact of bioactive compound lipophilicity and physiological ionic strength on the interaction between protein and bioactive compound. Lipophilicity influenced the strength of protein-bioactive compound interaction, while ionic strength changed the mode of interaction from static to static-dynamic quenching. The morphology of the nano and micro complexes formed with protein varied depending on the nature of encapsulated bioactive compound. Finally, bio-nano interaction involving giant unilamellar vesicles and curcumin-loaded pea protein of various surface functionalities as model biomembrane and nanoparticles respectively, was investigated. The result showed that while the protein/chitosan shell stabilizes bioactive compounds from degradation, the bioactive compound modulates their interaction with biomembrane. Overall, this work has demonstrated that for a rational design of protein-based nano/micro-encapsulation system, it is essential to consider the influence of the structural and physicochemical properties of proteins and bioactive compounds, stabilizing intermolecular forces, ionic strength of the environment, lipophilicity of the bioactive compounds, mechanism of release and modulation of cytotoxicity by bioactive compound. For instance, in high ionic strength solution, the stoichiometric ratio between protein carrier and bioactive compounds influences the stability of the complex. Balancing the intermolecular forces in the shell and core of bilayer complexes is essential for the stability of nanocomplexes and the presence of bioactive compound stabilizes the macromolecular carrier to minimal biomembrane disruption.

Lung cancer is the leading cause of cancer-related death worldwide. Exclusively found in citrus peels, the inhibitory effects of polymethoxyflavones (PMFs) on 3 human non-small cell lung cancer cells have been investigated. Results showed that monodemethylated PMFs at 5-position potently inhibited lung cancer cells than those of their permethoxylated counterparts. The inhibition of cancer cells caused by monodemethylated PMFs was associated with both extensive cell cycle arrest and apoptosis as a result of modulation of key oncogenic signaling proteins. Treatment with different bioactive compounds in combination may enhance inhibitory effects on lung cancer due to their synergistic interaction among these agents. Results showed that both nobiletin/atorvastatin (NBT/ATST) and tangeretin/atorvastatin (TAN/ATST) co-treatments at low doses exerted strong synergy as confirmed by isobologram analysis, and also produced much stronger inhibitory effects on lung cancer cells in comparison to those produced by NBT, TAN, or ATST alone at higher doses. Flow cytometry analysis showed both NBT/ATST and TAN/ATST co-treatments significantly induced cell cycle arrest and apoptosis, and these molecular events were involved with prenylation of RhoA which subsequently resulted in alteration of key signaling proteins. Supplementation of mevalonate or geranylgeranyl pyrophosphate significantly counteracted the effects caused by NBT/ATST. Inhibitory effects of metabolites of PMFs against lung cancer cells were significantly stronger than those produced by their parental compounds. Treatments of PMFs significantly inhibited lung tumorsphere formation and aldehyde dehydrogenase bright cells implicating the potential utilization of these compounds to target lung cancer stem cells.