O presente trabalho teve como objetivo realizar a extração de lignanas e outros compostos fenólicos do farelo de linhaça desengordurado e avaliar a atividade antifúngica dessas frações frente a fungos leveduriformes do gênero Candida. Obteve-se o extrato bruto através de maceração com solução hidroalcólica. O extrato bruto foi hidrolisado em condições básicas e ácidas, objetivando-se o isolamento de heterosídeos e agliconas, respecitvamete. A lignana secoisolariciresinol diglicosídeo foi quantificada por cromatografia líquida de alta eficiência. O flavonoide herbacetin diglicosídeo e a lignana anidrosecoisolariciresinol foram identificados por espectrometria de massas. A avaliação da atividade antifúngica foi realizada in vitro e foi observado que tanto o extrato bruto como o extrato obtido por hidrólise alcalina inibiram o desenvolvimento de Candida krusei CK 02 e Candida parapsilosis RL 20 na concentração de 500 μg/mL. O extrato obtido por hidrólise ácida, na mesma concentração, apresentou atividade inibitória frente a Candida krusei CK 02, Candida parapsilosis RL 20 e Candida tropicallis ATCC 750. A investigação das condições de hidrólise ácida, a melhor caracterização de seus componentes e a avaliação da ação antifúngica dos extratos frente a outros gêneros de fungos constituem perspectivas para trabalhos futuros. / The aim of the present work was carry out the extraction of lignans and other phenolic compounds from defatted flaxseed meal and evaluate the antifungal activity of these fractions against yeast fungi of Candida genus. The crude extract was obtained by maceration with a hydroalcoholic solution. The crude extract was hydrolyzed in basic and acidic conditions, aiming the isolation of glycosides and aglycones, respectively. Secoisolariciresinol diglucoside was quantified by highperformance liquid chromatography. The flavonoid herbacetin diglucoside and the lignan anhydrosecoisolariciresinol were identified by mass spectrometry. The avaliation of antifungal activity was performed in vitro and it was observed that both the crude extract and the extract obtained by alkaline hydrolysis inhibited the development of Candida krusei CK 02 and Candida parapsilosis RL 20 at a concentration of 500 ug/mL. The extract obtained by acid hydrolysis, in the same concentration, showed inhibitory activity against Candida krusei CK 02, Candida parapsilosis RL 20 and Candida tropicallis ATCC 750. The investigation of acid hydrolysis conditions, a better characterization of its components and the avaliation of antifungal activity against other fungi genera represents good potencial for future work.

O presente trabalho teve como objetivo realizar a extração de lignanas e outros compostos fenólicos do farelo de linhaça desengordurado e avaliar a atividade antifúngica dessas frações frente a fungos leveduriformes do gênero Candida. Obteve-se o extrato bruto através de maceração com solução hidroalcólica. O extrato bruto foi hidrolisado em condições básicas e ácidas, objetivando-se o isolamento de heterosídeos e agliconas, respecitvamete. A lignana secoisolariciresinol diglicosídeo foi quantificada por cromatografia líquida de alta eficiência. O flavonoide herbacetin diglicosídeo e a lignana anidrosecoisolariciresinol foram identificados por espectrometria de massas. A avaliação da atividade antifúngica foi realizada in vitro e foi observado que tanto o extrato bruto como o extrato obtido por hidrólise alcalina inibiram o desenvolvimento de Candida krusei CK 02 e Candida parapsilosis RL 20 na concentração de 500 μg/mL. O extrato obtido por hidrólise ácida, na mesma concentração, apresentou atividade inibitória frente a Candida krusei CK 02, Candida parapsilosis RL 20 e Candida tropicallis ATCC 750. A investigação das condições de hidrólise ácida, a melhor caracterização de seus componentes e a avaliação da ação antifúngica dos extratos frente a outros gêneros de fungos constituem perspectivas para trabalhos futuros. / The aim of the present work was carry out the extraction of lignans and other phenolic compounds from defatted flaxseed meal and evaluate the antifungal activity of these fractions against yeast fungi of Candida genus. The crude extract was obtained by maceration with a hydroalcoholic solution. The crude extract was hydrolyzed in basic and acidic conditions, aiming the isolation of glycosides and aglycones, respectively. Secoisolariciresinol diglucoside was quantified by highperformance liquid chromatography. The flavonoid herbacetin diglucoside and the lignan anhydrosecoisolariciresinol were identified by mass spectrometry. The avaliation of antifungal activity was performed in vitro and it was observed that both the crude extract and the extract obtained by alkaline hydrolysis inhibited the development of Candida krusei CK 02 and Candida parapsilosis RL 20 at a concentration of 500 ug/mL. The extract obtained by acid hydrolysis, in the same concentration, showed inhibitory activity against Candida krusei CK 02, Candida parapsilosis RL 20 and Candida tropicallis ATCC 750. The investigation of acid hydrolysis conditions, a better characterization of its components and the avaliation of antifungal activity against other fungi genera represents good potencial for future work.

Bast fibrous renewable materials, as commercial crops for the manufacture of textile based and other products, have been used for thousands of years to satisfy certain human needs, such as for shelter, clothing, source of energy and tools, and to sustain the livelihood of many communities in countries, such as in Asia, Europe and Africa. Concern for the environment has led to a number of global initiatives that favour the use of natural fibres. It is forecast that the global fibre demand by 2050 will be 130 million tonnes, which the cotton and synthetic fibre production will not be able to meet. Other than the environmental concerns, the renewed global interest in natural fibres, such as flax and hemp, forms part of a strategy to satisfy the fibre demand. South Africa, notwithstanding the fact that the flax and hemp industries have long been established for thousands of years in Europe, Asia and Egypt (Africa), has no history of the breeding, growing, processing and production of these cash crops. The renewed global interest in these crops has also stimulated interest from South Africa with respect to the development of a flax and hemp fibre industry so as to address some of the socio-economic challenges confronting the country today in its attempts to bring about real development in the rural areas through the cultivation and complete beneficiation of these fibre crops. In this regard, the relevant stakeholders, namely government, research councils, tertiary education institutions, farmers and communities worked together to investigate whether South Africa has the agronomic and climatic conditions, technical expertise as well as the necessary processing and production infrastructure to support the development of a local flax and hemp agro-industry. The purpose of this study was firstly to evaluate the performance of EU flax and hemp cultivars grown under different agronomic conditions in South Africa, and their effect on straw biomass yield, fibre yield and total fibre yield per hectare as well as associated fibre properties, and secondly to undertake mechanical fibre modification trials aimed at producing flax and hemp fibres with fibre diameters close to those of cotton. The minimum fibre diameter targeted being 20μm. In accordance with the project objectives and work plan, selected dew retted flax and hemp straw samples from the various planting sites selected by the Agriculture Research Council – Institute for Industrial Crops, representing all the agronomic conditions under investigation, were taken to the CSIR for fibre extraction and testing. A relatively inexpensive and easy to operate Russian designed and manufactured machine, the CMT-200M breaker-cum-scutcher, was used for fibre extraction and the resultant extracted fibre bundles were further refined, using a steel comb, to obtain optimal fibre separation before their physical and chemical properties were evaluated. Results obtained in this research study proved that the climatic and agronomic conditions in South Africa were suitable for the cultivation of flax and hemp, notwithstanding the fact that the fibre yields achieved for hemp were lower than the minimum criteria of 23 percent, and that for flax only just exceeded the 25 percent minimum. The lack of local technical expertise on the growing and retting of flax and hemp, contributed to the low fibre yields. The planting parameters which were found to produce good results for the cultivation of hemp were the October planting date, using a row spacing of between 12.5 to 25 cm, with a seeding density of between 80 – 100kg, and the application of 50 – 100kg nitrogen fertiliser. The use of extra artificial lighting and herbicide treatment did not appear to beneficially improve the hemp fibre yields. Similar considerations for flax cultivation in the Southern Cape region, particularly Oudtshoorn and Outeniqua, indicated that May to July planting dates, using inter-row spacing of 25cm and sowing density of 63kg seed.haˉ¹, produced a fibre yield and total fibre yield per hectare above the minimum values of 25 percent and 0,8 tonnes per hectare, respectively, quoted in the literature.

Two experiments determined the effects of flaxseed supplementation on reproductive parameters in sheep. In experiment one, 240 multiparous Rambouillet ewes were assigned to one of two treatments: basal ration alone or basal ration with a Flaxlic® Sheep Tub offered over 35 days. Serum was collected weekly for progesterone (P4). Flaxseed supplementation did not improve progesterone concentration or reproductive parameters (P ≥ 0.26). In experiment two, 120 Rambouillet ram lambs were assigned to one of two treatments: basal ration alone or basal ration with a Flaxlic® Sheep Tub offered over 112 days. Scrotal circumference measurements, serum for testosterone, and semen were collected on day 84 and 112. Day effects were found for select semen measurements, testosterone, weight, and scrotal circumference (P ≤ 0.05). There was no effect of treatment on testosterone concentration (P = 0.99) or any semen quality characteristics (P ≥ 0.33). Overall, tub supplementation did not alter reproductive ability.

Ovarian cancer is a deadly gynecological disease that ranks fifth in all cancer-related deaths inwomen. The disease is often detected at an advanced stage and lack of sensitive predictive biomarkerscontribute to its poor prognosis, which is also responsible for the current five-year survival rate of only47%. Our laboratory has previously shown that a whole flaxseed supplemented diet decreases the onsetand severity of ovarian cancer in the laying hen, the only known animal model of spontaneous ovariancancer. Flaxseed is rich in omega-3 fatty acids (OM3FA), mostly α-Linoleic acid (ALA), which getsconverted to Docosahexaenoic acid (DHA) by the action of delta-6 desaturase enzyme. We have also shownthat the flaxseed diet in hens induces CYP1A1 expression in the liver while suppressing both CYP1B1 andCYP3A4 expressions. The upregulation of CYP1A1 parallels the increase in 2-hydroxyestradiol and the 2-methoxyestradiol (2MeOE2) level in the serum of the chickens. Flaxseed diet induces apoptosis to ovariantumors and not in the normal ovarian tissues. The current work explores the pro-apoptotic actions of oneof the biologically derived compounds of flaxseed diet, 2MeOE2, and describes one of its novel molecularactions. Our results indicate that 2MeOE2 -mediated pro-apoptotic actions are partly dependent on thecatalytic activation of protein kinase C delta (PKCd), which is responsible for key apoptotic histonemodifications and p38 MAPK (MAPK14) phosphorylation. Phosphorylated MAPK14, in turn, results inmore caspase-3 cleavage which activates more PKCd, therefore amplifying the apoptotic signal.The other part of this work explains one of the key anti-cancer actions of a flaxseed diet. Flaxseedreduces the number of endothelial cells and increases pericytes to endothelial cell ratio in ovarian tumorscompared to control diet-fed birds, indicating a reduction in tumor angiogenesis and an improvement inblood vessel maturation. Flaxseed decreases a-smooth muscle actin expression in ovarian tumors, whichmarks a decrease in cancer-associated fibroblasts (CAFs) in the tumor stroma. Flaxseed also reduces fibrosisin ovarian tumors. Flax -mediated reduction of ovarian fibrosis parallels an increased infiltration ofimmune cells in the preneoplastic ovaries and ovarian tumors. Therefore, this work describes how the flaxdiet differentially targets the ovarian tumor cells and the tumor stroma in preventing the incidence andseverity of ovarian cancer. Dietary supplementation with flaxseed can, therefore, be a healthy lifestylechoice as well as a potential adjuvant therapy for immunotherapy-based treatment modalities.

Ovarian cancer is the fifth most lethal cancer in women (1) and the most lethal gynecological malignancy. In 2018, there were approximately 22,240 new diagnosed cases of ovarian cancer and 14,070 deaths in the United States alone (2). The lifetime risk for developing ovarian cancer in the United States is 1.3% or approximately 1 in 78 women. The five-year survival rate for women with ovarian cancer is a grim 47.6% (2) while the average five year survival rate for all cancers is about 68%. This dismal prognosis for ovarian cancer patients indicates the critical need for improved treatment options, efficient early detection methods and effective preventative measures for ovarian cancer (1). The objective of this study was to determine if DHA causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of NF-κB regulated transcription in the ovary. DHA is a 22 carbon long-chain omega-3 polyunsaturated fatty acid that is biologically derived from Alpha-linolenic acid (ALA) found in flaxseed. COX-2 is an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins. Prostaglandin E2 (PGE2) is a key regulator of inflammation which has been shown to be highly associated with ovarian cancer development and progression. Our laboratory studies ovarian cancer in the laying hen because it is the only known animal model to naturally develop ovarian cancer that both pathologically and histologically matches that of the human form of the disease. Dietary flaxseed is one of the richest vegetable sources of omega-3 polyunsaturated fatty acids. Our previous studies have shown that in laying hens, a long-term flaxseed supplemented diet reduces the incidence and severity of ovarian cancer and decreases COX-2 and PGE2. It was hypothesized that DHA, derived from ALA found in flaxseed, decreases inflammation in the ovaries by suppressing the activation of COX-2 and the production of PGE2 through inhibition of the NF-κB pathway. For this study, an NF-κB reporter plasmid was transfected into HEK293 cells. The reporter plasmid (“met-luc”) produces a secreted luciferase allowing sequential analysis of media from DHA and TNF-α treated cells to assess changes in NF-κB transcriptional activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB was used as a positive control. NF-κB activation was also assessed by measuring its nuclear translocation and cytoplasmic accumulation through immunocytochemistry (ICC) and western blot analysis. In a parallel study, immortalized ovarian surface epithelial (IOSE) cells were challenged with the same treatments of DHA and TNF-α. In these cells, COX-2 mRNA was assessed through RT-qPCR and COX-2 protein expression was analyzed through ICC and western blot.Our results indicate that DHA acts in a cell specific manner to reduce inflammation associated with cancer. We have found that in HEK293 cells DHA reduces TNFα induced NF-κB reporter activity. In contrast, ALA does not affect NF-κB reporter activity. HEK293 cells treated with TNFα alone indicated a dose-dependent increasing trend in nuclear translocation of the NF-κB p65 subunit and a decreasing trend in cytoplasmic p65, suggesting potential increased pathway activation. ICC suggests DHA treatment causes increased cytoplasmic sequestration of the NF-κB p65 subunits indicating inhibition of TNFα induced NF-κB activation. Western blot data also indicates a decreasing trend in nuclear NFκB p65 when cells are pretreated with DHA and subsequently challenged with TNF. The IOSE cells, were the only cells out of the cell lines tested (BG1, HEYC2, TOV112D, SKOV3, HEK293) to express COX-2. In these IOSE cells, TNFα alone showed a dose-dependent increasing trend in COX-2 protein (analyzed through ICC and western blot) and mRNA levels (analyzed through RT-qPCR). ICC analysis revealed that DHA reduces TNF induced COX-2 protein expression. However, the western blot did not further support this observation. Only a slight non-significant reduction with DHA treatment was observed. In addition, both DHA and TNFα, while also not significant, seemed to increase mRNA levels of COX-2 compared to control. This slight decreasing trend in COX-2 protein expression and increase in mRNA, could indicate a possible post-transcriptional mechanism of regulation of COX-2 by DHA independent of NF-κB in the IOSE cells. These data suggest that DHA could act via distinct mechanisms in a cell specific manner to potentially reduce COX-2 and subsequently PGE2 levels. DHA can act at the transcriptional level by reducing the nuclear translocation of NF-κB and transcriptional activation of NF-κB target genes such as COX-2 in some cell types. DHA also has the potential to work via a post-transcriptional mechanism to inhibit COX-2 and in turn reduce PGE2 levels. Both mechanisms ultimately have the potential to decrease the inflammation associated with ovarian cancer. This study describes the anti-inflammatory action of dietary flaxseed consumption, making flaxseed supplementation a promising preventive measure for reducing the risk of ovarian carcinogenesis.

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