Avaliação in vitro do potencial anti-lipogênico e antiinflamatório de extrato biotransformado de resíduo de cítricos

Orientador: Juliana Alves MacedoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: A obesidade é caracterizada pelo excesso de tecido adiposo com aumento na infiltração de macrófagos, e secreção de citocinas pró-inflamatórias, levando a um estado inflamatório subclínico crônico. Diversos produtos têm sido avaliados na tentativa de prevenir e tratar a obesidade e suas complicações. Dentre os compostos estudados, destaca-se a importância dos fenólicos. As frutas cítricas são importante fonte de flavonoides, com destaque a casca, que apresenta teor mais elevado que a polpa. Assim, este estudo objetivou avaliar o potencial anti-lipogênico e anti-inflamatório de extratos de flavonoides de cítricos. Os extratos foram obtidos a partir do resíduo industrial de frutas cítricas, modificados por bioprocesso fermentativo (extrato "Biotransformado"), e dois extratos controles, o primeiro sem processamento ("In Natura"), e o segundo com o processamento térmico equivalente ao sofrido pelo extrato fermentado (extrato "Autoclavado"). Realizaram-se estudos in vitro para determinar atividade antioxidante, ação na lipogênese e lipólise em células 3T3-L1, e o efeito anti-inflamatório em células RAW264.7 e 3T3-L1. O extrato do resíduo de cítricos obtido após a biotransformação apresentou-se como boa fonte de hesperitina e naringenina, flavanonas encontradas em baixa quantidade na natureza. O extrato "Biotransformado" apresentou menor quantidade de flavanonas totais, no entanto, a capacidade antioxidante foi semelhante de acordo com DPPH e ORAC, indicando um novo perfil fenólico com maior potencial bioativo que o original. Os extratos apresentaram baixa citotoxicidade nas concentrações entre 0,01-1,00 mg/mL. As amostras não apresentaram muita influência nos processos de diferenciação dos adipócitos a partir de fibroblastos, porém a adição de extrato no meio de maturação causou uma diminuição dose dependente na acumulação de lipídeos das células, com reduções entre 22% e 48% para os extratos "Biotransformado" e "In Natura". O extrato "Biotransformado" foi o único que apresentou efeito na liberação de glicerol livre no sobrenadante da cultura (2,39 ± 0,17 - 5,24 ± 0,29 mg/mL de glicerol), indicando seu papel na lipólise. A adição dos extratos em macrófagos (RAW264.7) causou menor secreção de indicadores inflamatórios, como TNF-? e NO, com melhores resultados para o extrato "Biotransformado". A adição dos extratos também causou menor expressão proteica do fator de transcrição NF?B. Em co-cultura de RAW264.7 e 3T3-L1, o tratamento com 1,0 mg/mL de extratos "Biotrasformado" e "In Natura" reduziu a secreção de TNF-? (30,7% e 14,9%) e IL-6 (43,4% e 42,7%) em relação ao Controle sem tratamento. Ainda, o extrato "Biotransformado" a uma concentração de 1,0 mg/ml promoveu maior aumento de adiponectina em relação ao "In Natura" (66,0% e 35,3%, respectivamente). Quando a co-cultura recebeu estímulo com LPS, a adição do extrato reduziu a concentração de IL-6 e TNF-? e causou maior aumento na concentração de adiponectina. A biotransformação dos compostos fenólicos do extrato de resíduo de cítricos foi capaz de modificar o perfil de flavanonas, aumentando as agliconas. Ainda, o extrato demonstrou potencial para uso na indução da lipólise e atividade anti-inflamatória em cultura de macrófagos e em co-cultura de macrófagos/adipócitos. Assim, podemos concluir que o bioprocessamento pode contribuir para o desenvolvimento de um produto com potencial uso no tratamento da obesidade e da inflamação associadaAbstract: Obesity is characterized by excess adipose tissue with increased macrophage infiltration and pro-inflammatory cytokines secretion, leading to a subclinical chronic inflammatory state. Many food products have been evaluated in an attempt to prevent and treat obesity and its complications. Among the compounds studied, phenolics are of great interest. Citrus fruits are an important source of flavonoids, especially citrus peel, which present higher content in relation to pulp. Thus, this study aimed to evaluate anti-lipogenic and anti-inflammatory potential of a citrus flavonoid extract. The extracts were obtained from industrial residue of citrus fruits, modified by fermentative bioprocess ("Biotransformed" extract) and two control extracts, the first one without any processing ("In Natura"), and the second with the equivalent thermal process suffered by fermented extract ("Autoclaved"). For this purpose, in vitro assays were performed to determine their antioxidant activity, lipogenesis and lipolysis activity in 3T3-L1 cell line, and anti-inflammatory effect on RAW264.7 and 3T3-L1.The citrus residue extract obtained after "Biotransformation" was a good source of hesperitin and naringenin, flavanones often found in low quantity in nature. Biotransformed residue presented smaller amount of total flavanones, however the antioxidant capacity of the extracts was similar according to DPPH and ORAC assays, indicating a new phenolic profile with greater bioactive potential than the original. The extracts showed low cytotoxicity in concentrations ranging from 0.01-1.00 mg / mL, in the cells of interest. Samples did not have much influence in the new adipocytes differentiation processes from fibroblasts, on the other hand, the addition of the extracts in maturation medium in adipocytes caused a dose-dependent decrease in lipid accumulation, reaching a diminution of 22% and 48% for Biotransformed and In Natura extracts. Biotransformed extract was the only that presented some effect on glycerol release (2.39 ± 0.17 - 5.24 ± 0.29 mg/mL of glycerol), indicating its role in lipolysis. Treatment of RAW 264.7 cell with extracts caused lower secretion of inflammatory indicators, as TNF-? and NO, with greater results for "Biotransformed" extract. There was also lower protein expression of NF?B with the treatment. In RAW264.7 and 3T3-L1 co-culture, treatment with 1.0mg/mL of "Biotrasformed" and "In Natura" extracts reduced secretion of TNF-? (30.7% and 14.9%) and IL-6 (43.4% and 42.7%) compared to Control without any treatment. Still, Biotransformed extract at a concentration of 1.0mg/mL promoted greater increase in adiponectin in relation to In Natura (66.0% and 35.3% respectively). When the co-culture received LPS stimulus, addition of the extract reduced IL-6 and TNF-? concentration and caused a greater increase in adiponectin. Biotransformation of phenolic compounds from citrus extract residue was able to modify flavanones profile, increasing the aglicones. Still, the extract showed potential for use in inducing lipolysis and anti-inflammatory activity in macrophages culture and co-culture of macrophages with adipocytes. Thus, we can conclude that bioprocess can contribute to the development of a product with potential use in treatment of obesity and associated inflammationDoutoradoNutrição Experimental e Aplicada à Tecnologia de AlimentosDoutora em Alimentos e Nutrição2015/04555-2FAPES

Ethanol Increases NADPH Oxidase-derived Oxidative Stress and Induces Apoptosis in Human Liver Adenocarcinoma Cells (SK-HEP-1)

Alcohol-induced liver injury is linked to oxidative stress and increased production of reactive oxygen species (ROS). Oxidative stress is an early event in the process of apoptosis. However, it is not completely understood how ethanol-induced oxidative stress induces apoptosis. In contrast, nicotinamide adenine dinucleotide phosphate oxidase (NOX) is known to generate ROS in hepatocytes. The purpose of the present study was to determine whether or not ethanol-induced ROS generation stimulates the death receptor or mitochondrial pathways of apoptosis in alcohol dehydrogenase containing human liver adenocarcinoma (SK-HEP-1) cells. Treatment with ethanol increased the generation of ROS and expression of NOX4 mRNA, and also induced mitochondrial dysfunction in SK-HEP-1 cells. Moreover, ethanol induced the activation of caspase-8 and -3 in hepatocytes. These activities were suppressed by pretreatment with N-acetyl-cysteine, an antioxidant, or apocynin, an inhibitor of NOX activity. These results suggested that ethanol induces an increase in NOX-derived ROS generation upstream of caspase-8 activation and in the mitochondria in SK-HEP-1 cells. In conclusion, this study demonstrated that ethanol increases the generation of ROS and subsequently induces apoptosis using a mechanism involving mitochondrial dysfunction and caspase activation in SK-HEP-1 cells

Lymph Drainage during Wound Healing in a Hindlimb Lymphedema Mouse Model

Background: Although lymphedematous skin exhibits delayed wound healing, little is known about lymph drainage during wound healing. We investigated the wound healing process in the presence of lymphatic dysfunction. Methods and Results: The right inguinal lymph nodes (iLNs) and the surrounding tissue were excised in each mouse (the operation side), and a sham operation was performed in the left hindlimb (the control side). The next day, full-thickness wounds were made on both hindlimbs. The right hindlimb exhibited acute edema until day 3; however, it started to improve after day 4, and the wound area and epithelialization ratio were similar on both sides. Indocyanine green (ICG) was injected into both hindlimbs to observe lymph flow. On the operation side, ICG leaked out of the surgical site or remained at the injection site until day 2. Some lymph flow toward the existing lymph vessels was seen on day 3, and on day 10, lymph flow toward the axial LNs was detected on the operation side in all mice. On the operation side, the number of dermal lymph vessels was significantly increased on days 3 and 15. The dermal lymph vessel area of the peripheral wound was significantly smaller on the operation side. Conclusions: In a hindlimb lymphedema mouse model, lymph transiently accumulated in subcutaneous tissue, and then was gradually absorbed by the existing lymph vessels. The increase in the number of lymph vessels contributes to lymph drainage during wound healing. Acute lymphedema because of transient lymphatic dysfunction has little effect on wound healing. © Copyright 2017, Mary Ann Liebert, Inc. 2017.Embargo Period 12 month

The Relationship between Cutaneous Wounds Made on Obese Mice or Those with Decreased Body Weight and Serum Leptin Level

Purpose: Not all obese people have hyperglycemia. We wondered about the healing progress in obese people without hyperglycemia. The purpose of this study is to observe the cutaneous wound healing process. Methods: Three-week-old male mice were fed high-fat diets (containing 60% fat) in the diet group, and commercial diets in the control group, ad libitum for 15 weeks. Circle-full-thickness cutaneous wounds were made on the dorsal skin of mice. From day 0 to day 15 after wounding, we analyzed wound healing process. We measured the blood concentration of leptin, and observed the distribution of leptin-positive cells in each wound. Results: Mean body weight, the areas of subcutaneous fat and visceral fat, and the weight of epididymal fat in the diet group were significantly greater than those in the control group at 15 weeks after feeding. The diet group did not feed on the diet after wounding; their body weight decreased remarkably to the level of the control group. The ratio of wound area, re-epithelialization, and collagen fibers did not differ between the diet and control groups on each day. The blood concentration of leptin in the diet group was significantly greater than that in the control group before wounding and until day 6 after wounding (day 0, 10 hour and day 1: p < 0.01, day 6: p < 0.05). Conclusion: The results show that the wound healing process is similar between obese and non-obese mice, and that the decrease in the leptin level in the obese mouse to that in the non-obese mouse may depend on the decrease of body weight of obese mouse

部分加水分解した羊毛繊維および羊毛粉末のサクシニル化とその吸水性への効果

Wool fiber and powder were succinylated with succinic anhydride in aqueous alkaline solution in order to improve water absorbability after partial hydrolysis with HCl that was a pretreatment to enhance the reactivity. Succinylation after partial hydrolysis raised the water absorbabilities of both samples to ca. 5 times. The water absorbabilitiy of the succinylated samples and add-ons of succinic anhydride showed a linear relationship in each case of wool fiber and powder, which suggests that carboxyl groups introduced in the samples by succinylation contribute the absorbance of water. Water regain of these samples also increased by 1.0～4.1% after the partial hydrolysis and succinylation

The effect of isoflavone-daidzein oral medication on cutaneous wound healing in female ovariectomized mice

This study investigated the influence of oral administration of isoflavone-daidzein on the cutaneous wound healing process in female ovariectomized mice. Eight-week-old female mice were divided into groups of ovariectomized mice and mice administered daidzein after an ovariectomy. Two full-thickness wounds on the dorsum were made in mice in both groups. There was no significant difference between wound areas of the two groups from wounding to healing during 15 days. The area in the group administered daidzein tended to be smaller than that in the ovariectomized group during the inflammatory phase 4 and 5 days after wounding . The rate of re-epithelialization in the group administered daidzein tended to be higher than that in the ovariectomized group in the inflammatory phase on day 3 (40.7 ± 17.6% and 21.0 ± 16.8%, respectively). Therefore, the administration of daidzein under lack of estrogen is expected to reduce the inflammation period and promote re-epithelialization. この研究は、卵巣摘出した雌マウスに皮膚創傷を作製し、経口投与したイソフラボン の一種であるダイゼインが、創傷治癒にどのような影響を与えるかを観察したもので ある。8 週令の雌マウスを卵巣摘出群と卵巣摘出し創作製した後にダイゼインを与え た2 群に分けた。両群共、卵巣摘出後、ダイゼインを含まない精製飼料で２週間飼育 後に、左右の背部に直径4mm の皮膚全層欠損層を作製した。創作製後、ダイゼインを 含まない飼料とダイゼインを含む飼料で2 週間飼育した。ダイゼインは、飼料1g に 0.01mg 含むように作製した。両群の創面積は、2 週間の間の毎日において、有意差は 見られなかった。しかし、炎症期である創作製後4 と5 日では、ダイゼイン食で飼育 した群が無ダイゼイン食で飼育した群が、やや創面積が小さい傾向がみられた（それ ぞれ、p 値が0.061、0.083 であった）。創作製後の3 日での、再上皮化の割合は、ダイゼイン群で40.7 ± 17.6%、無ダイゼイン群で21.0 ± 16.8%となり、ダイゼイ ン群はより上皮化が進んでいる傾向が見られた (p = 0.07)。これらの結果は、エス トロゲン欠乏状態で、ダイゼインの経口投与が、創傷治癒において、炎症を抑制し上 皮化を促進することを示唆している