Advances in the biosynthesis and application of bixin / 生物工程学报

Since synthetic pigments are potentially harmful to human health, natural ones such as bixin, one of the carotenoids, are favored. As the second widely used natural pigment in the world, there is significant interest in the biosynthetic pathway of bixin which has not been fully elucidated. This review summarizes the chemical properties, extraction methods, biosynthetic pathway and application of bixin. In addition, we compared the difference between traditional extraction methods and new extraction techniques. Moreover, we described the genes involved in the biosynthetic pathway of bixin and the effects of abiotic stress on the biosynthesis of bixin, and discussed the application of bixin in food, pharmaceutical and chemical industries. However, the researches on bixin biosynthesis pathway are mostly carried out at the transcriptome level and most of the gene functions have not been elucidated. Therefore, we propose to characterize the entire bixin biosynthetic pathway using techniques of genomics, bioinformatics, and phytochemistry. This will help facilitate the synthetic biology research of bixin and development of bixin into new drugs.

Desenvolvimento e avaliação de nova preparação de anfotericina B e/ou cetoconazol em dispersão de bixina, pullulan e trealose / Development and evaluation of novel preparation of amphotericin B and/or ketoconazole in dispersion of bixin, pullulan and trehalose

Muitos pacientes acometidos por infecções fúngicas sucumbem devido a não eficácia dos antibióticos ou por toxicidade dos mesmos. Anfotericina B é um dos antifúngicos mais eficientes do mercado apesar de sua alta toxicidade, tem estrutura poliênica e é um composto insolúvel em água, sendo necessário o uso de adjuvantes e novas tecnologias para preparo de formulações eficazes. Cetoconazol é um composto imidazólico, também com ação antifúngica de grande espectro de ação e difícil solubilização em meio aquouso, porém solúvel somente em baixos valores de pH. Estudos têm demonstrado a utilização de bixina na preparação de dispersões aquosas de compostos insolúveis ou pouco solúveis em água. Bixina é o principal composto das cascas de semente de Bixa orellana (urucum), sendo um carotenoide insolúvel em água, porém, permite preparações na forma de nanodispersões aquosas com incorporação de fármacos apolares ou lipofílicos. O objetivo deste trabalho foi preparar anfotericina B e cetoconazol na forma de nanodispersões a partir de bixina, utilizando pullulan e trealose como adjuvantes e avaliar estabilidade e eficácia antimicrobiana por ensaios físico-químicos e microbiológicos. Pullulan é um polissacarídeo constituído por unidades de maltotriose, com propriedades adesivas e capacidade de formar biofilmes, enquanto trealose é um composto com duas unidades de glicose, com boa estabilidade em faixas de pH de 3 a 10 e capaz de suportar altas temperaturas, como processos de esterilização por calor úmido. Ensaios físico-químicos demonstraram boa manutenção das características das preparações propostas neste projeto, como, por exemplo, diâmetro hidrodinâmico e potencial zeta das estruturas das nanodispersões de bixina e antifúngicos e também eficácia antimicrobiana frente a Candida albicans ATCC 10231. Os resultados apresentam perspectivas para aprimoramentos de formulações com fármacos pouco solúveis ou insolúveis em água, pesquisa de novos biomateriais e outras aplicações nas áreas farmacêutica e cosmética

Many patients with fungal infections succumb due to ineffectiveness or toxicity of antibiotics. Amphotericin B is one of the most efficient antifungals on the market despite its high toxicity. It presents polyenic structure and is a water-insoluble compound. In this case, it is necessary to use adjuvants and new technologies to prepare effective formulations. Ketoconazole is an imidazolic compound, also with broad spectrum antifungal action and difficult solubilization in aqueous medium but it is soluble at low pH values. Studies have demonstrated the use of bixin in the preparation of aqueous dispersions of insoluble or poorly soluble compounds in water. Bixin is the main compound of Bixa orellana (annatto) seed husks, being a water-insoluble carotenoid, but it allows preparations in the form of aqueous nanodispersions with incorporation of apolar or lipophilic drugs. The objective of this work was to prepare amphotericin B and ketoconazole as nanodispersions from bixin, using pullulan and trehalose as adjuvants and to evaluate them under aspects of stability and efficacy by physicochemical and microbiological assays. Pullulan is a polysaccharide consisting of maltotriose units with adhesive properties and ability to form biofilms, while trehalose is a compound with two glucose units with good stability at pH ranges from 3 to 10 and capable of withstanding high temperatures such as processes of sterilization by moist heat. Physicochemical tests demonstrated good maintenance of the characteristics of the preparations proposed in this project, such as hydrodynamic diameter and zeta potential of bixin and antifungal nanodispersions and also antimicrobial efficacy against Candida albicans ATCC 10231. The results present prospects for improvement. of poorly soluble or water-insoluble drug formulations, research on new biomaterials and other applications in the pharmaceutical and cosmetic fields

The effect of the carotenoid bixin and annatto seeds on hematological markers and nephrotoxicity in rats subjected to chronic treatment with cisplatin

ABSTRACT This study assessed the protective effect of the carotenoid bixin and annatto seeds against possible nephrotoxicity induced with a single peritoneal administration of pharmacological cisplatin in male Wistar rats. After 48 h, the blood cell differential count showed a significant reduction in neutrophil counts in rats that received a diet rich in bixin when compared to the group that received only cisplatin. The use of cisplatin led to an increase in kidney weight. The carotenoid bixin attenuated renal injury, characterized by increased polymorphonuclear infiltration. No protective effect was observed with respect to Annatto. These results demonstrate the role of toxic cisplatin and suggest that bixin affords a protective effect against cisplatin-induced nephrotoxicity in adult Wistar rats.

Effects of bixin in high-fat diet-fed-induced fatty liver in C57BL/6J mice

Objective To evaluate the anti-obesity activity of bixin (BIX) on C57BL/6J mice which were fed a high-fat diet (HFD) and to determine the mechanism of this effect. Methods C57BL/6J mice were separately fed a high-calorie diet or a normal diet for 8 weeks, then they were treated with BIX for another 13 weeks. After administration for 13 weeks, the animals were sacrificed. Body adiposity, serum lipid level, and insulin resistance were evaluated. In addition, a histological assay of pancreas and liver, an evaluation of the inhibitory properties on pancreatic lipase, and α-amylase were conducted. Results Administration of BIX significantly decreased the body weight gain, adipocyte size, fat pad weights, hepatic lipid levels in HFD-induced obese mice. In addition, reduced liver weight exhibited decreased serum leptin levels, malic enzyme, glucose-6-phosphate dehydrogenase, hepatic fatty acid synthase, aspartate aminotransferase, alanine aminotransferase and hepatic phosphatidate phosphohydrolase activity. However, superoxide dismutase, catalase, glutathione peroxidase, and glutathione levels were increased in hepatic tissue. BIX also decreased lipid and carbohydrates absorption due to inhibition of pancreatic lipase and α-amylase. Long term supplementation of BIX significantly decreased hyperlipidemia, insulin resistance and glucose level. Decreased levels of hepatic steatosis and the islets of Langerhans appeared less shrunken in HFD-fed mice. Conclusions The antiobesity effect of BIX appears to be associated at least in part, to its inhibitory effect on lipids and carbohydrate digestion enzymes such as pancreatic lipase, α-glucosidase, and α-amylase. The results suggested that BIX also act as an antioxidant and may treat visceral obesity normalizing glucose levels, improving insulin resistance and increasing energy expenditure. Therefore, achiote which has a main component, the carotenoid BIX, could be a viable food for the treatment of obesity and diabetes.

Analysis of water soluble polysaccharides as a potential chemotaxonomic marker for landraces in Bixa orellana.

Annatto tree (Bixa orellana L.) is native to Brazil and is now under cultivation in many parts of world for its reddish orange ‘annatto’ dye. There are three types of landraces in annatto and they are distinguished based on fruit shape i.e., ovate, conical and hemispherical, whose pigment yield differs. Since annatto pigment yield varies with landrace, it is necessary to characterize markers towards the identification of landraces. In this study, we characterized water soluble polysaccharides (WSP) of twigs from three landraces using size-exclusion chromatography (SEC), fourier-transform Infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR) and gas liquid chromatography (GLC) for their potential use as chemotaxonomic markers to distinguish the landraces. GLC analysis on WSP showed hemispherical type contained 38% rhamnose, while conical and ovate types contained 17% and 34% glucose, respectively. Thus, glucose and rhamnose content of WSP could be used to distinguish the three landraces. Further, differences in calculated molecular weight as revealed by SEC (281.8, 151.3 and 79.4 kDa for conical, hemispherical and ovate types, respectively) could also be used to distinguish the three landraces.

Protective effect of bixin on carbon tetrachloride-induced hepatotoxicity in rats

BACKGROUND: The liver is an important organ for its ability to transform xenobiotics, making the liver tissue a prime target for toxic substances. The carotenoid bixin present in annatto is an antioxidant that can protect cells and tissues against the deleterious effects of free radicals. In this study, we evaluated the protective effect of bixin on liver damage induced by carbon tetrachloride (CCl4) in rats. RESULTS: The animals were divided into four groups with six rats in each group. CCl4 (0.125 mL kg-1 body wt.) was injected intraperitoneally, and bixin (5.0 mg kg-1 body wt.) was given by gavage 7 days before the CCl4 injection. Bixin prevented the liver damage caused by CCl4, as noted by the significant decrease in serum aminotransferases release. Bixin protected the liver against the oxidizing effects of CCl4 by preventing a decrease in glutathione reductase activity and the levels of reduced glutathione and NADPH. The peroxidation of membrane lipids and histopathological damage of the liver was significantly prevented by bixin treatment. CONCLUSION: Therefore, we can conclude that the protective effect of bixin against hepatotoxicity induced by CCl4 is related to the antioxidant activity of the compound.

Indução do sistema citocromo P450 em linhagens de hepatoma humano para utilização como modelo in vitro no desenvolvimento de fármacos / Induction of cytochrome P450 system in human hepatoma cell lines for using as in vitro model in drug development

Na etapa inicial do desenvolvimento de novos fármacos, a avaliação do metabolismo e da toxicidade é fundamental para definir seu potencial emprego como candidato a fármaco. Nestes estudos, diversos modelos in vitro são empregados, dentre eles linhagens de hepatoma humano. Entretanto, uma grande limitação ao uso deste modelo in vitro é a baixa expressão das enzimas do sistema citocromo P450. O carotenóide bixina, componente majoritário do anato (urucum), apresentou em estudos in vivo, a capacidade de induzir algumas isoformas do sistema citocromo P450, com a vantagem de apresentar baixa toxicidade. Neste trabalho, a fração lipossolúvel do anato (bixina) e hidrossolúvel (norbixina) foram avaliadas como indutores do sistema citocromo P450 em linhagens de hepatoma humano. Ensaios de MTT, empregando as linhagens HepG2, C3A e SK-HEP-1 indicaram que bixina e norbixina em concentrações abaixo de 0,22 mM são seguras quanto à citotoxicidade. A expressão dos genes CYP 1A1, 1A2, 2C9, 2B6, 2E1 e 3A4 foi avaliada, através de ensaios de RT-PCR em tempo real, em linhagens de hepatoma humano submetidas a tratamento com os compostos bixina e norbixina. Os resultados mostraram que células HepG2 e C3A tratadas com bixina nas concentrações de 0,05 e 0,1 mM, por períodos de 24 e 48 horas, apresentaram aumento de expressão da CYP 1A1 e CYP 1A2. Porém, a exposição de células HepG2 e C3A ao composto norbixina não resultou em aumento de expressão das isoformas avaliadas neste estudo. Os resultados deste trabalho indicaram o potencial emprego de bixina como agente indutor das CYPs 1A1 e 1A2, em linhagens de hepatoma humano utilizadas como modelo in vitro, para estudo de compostos cuja metabolização envolva uma destas vias, entretanto, estudos adicionais são fundamentais, a fim de avaliar a ação deste composto sobre outras isoformas do sistema citocromo P-450, bem como outros sistemas enzimáticos.

In the early development stage of the new drugs, the pharmacological and toxicological properties are critical to define the potential use of the candidate drug. During this stage, several in vitro models systems are employed, including human hepatoma cell lines. However, the main limitation of the use of cell lines as in vitro model is the low expression level of cytochrome P450 enzymes. A carotenoid knowed as bixin, the main pigment in the annatto (urucum), it has been reported to induce some isoforms of cytochrome P450 in rats, with the advantage of its low toxicity. In this work, the oil-soluble (bixin) and aqueous soluble extracts (norbixin) were evaluated as inducers of the cytochrome P450 system in human hepatoma cell lines (HepG2, C3A, SK-HEP-1). The results of MTT assays showed that bixin concentrations below 0.22 mM were not cytotoxic in HepG2, C3A and SK-HEP-1 cell lines. Expression changes in CYP 1A1, 1A2, 2C9, 2B6, 2E1 and 3A4 were evaluated, by real time RT-PCR and the results showed that the exposition to 0,05 mM and 0,1 mM bixin, for 24 and 48 hours of treatment, lead to an increase in CYP 1A1 and CYP 1A2 expression level. By contrast, the cytochrome P450 isoforms were not affected by the exposition to norbixin. In conclusion, this work indicated the potential use of bixin induced hepatoma cell lines as in vitro model for studies of biotransformation and toxicity of drugs involving CYP 1A, however, further studies are necessary to evaluate the effect of bixin on the other cytochrome P450 isoforms as well as other enzymatic systems.

Fatores que influenciam a reação de saponificação dos carotenóides presentes no urucum (Bixa orellana L. ) / Factors that influence saponification reaction in annatto (Bixa orellana L. ) carotenoids

Conduziu-se este trabalho, com o objetivo de estudar o efeito de diferentes concentrações de KOH (0,6 por cento, 1 por cento, 2 por cento, 3 por cento, 4 por cento e 5 por cento) e temperatura de extração (25ºC, 50°C, 60°C e 70°C) na reação de saponificação dos principais pigmentos de urucum. O progresso da reação foi acompanhado pela quantificação do teor de bixina e de norbixina por meio de cromatografia líquida de alta eficiência (CLAE). A maior concentração de base propiciou a maior conversão de bixina em norbixina (KOH 5 por cento - bixina: 0,44 g.100g-1, norbixina: 1,43 g.100g-1) e com a menor concentração não ocorreu conversão (KOH 0,6 por cento - bixina: 2,00 g.100g-1, norbixina: não detectada). A elevação da temperatura de extração esteve associada à redução do teor de bixina no meio e ao aumento do teor de norbixina (T70°C - bixina: 0,01 g.100g-1, norbixina: 2,34 g.100g-1; T25ªC - bixina: 1,04 g.100g-1, norbixina: 0,78 g.100g-1). Sendo assim, para extração de bixina seria recomendado o uso de soluções alcalinas pouco concentradas, ao passo que para a obtenção do pigmento hidrossolúvel são aconselhadas concentrações mais elevadas, podendo estar associadas a aquecimento.

This research aimed to evaluate the effect of different KOH concentrations (0.6 percent, 1 percent, 2 percent, 3 percent, 4 percent and 5 percent) and extraction temperature (25ºC, 50°C, 60°C and 70°C) on the progress of saponification reaction in annatto pigments. The progress of the reaction was monitored by quantification of bixin and norbixin using high performance liquid chromatography (HPLC). The highest alkali concentration made possible the maximum conversion of bixin into norbixin (KOH 5 percent - bixin: 0.44 g.100g-1, norbixin: 1.43 g.100g-1) and using the lowest concentration, saponification didn't take place (KOH 0.6 percent - bixin: 2.00 g.100g-1, norbixin: not detected). The elevation of extraction temperature was associated to a decrease of bixin content and to an increase of norbixin content (T70°C - bixin: 0.01 g.100g-1, norbixin: 2.34 g.100g-1; T25ºC - bixin: 1.04 g.100g-1, norbixin: 0.78 g.100g-1). Therefore, for the extraction of bixin is recommended to use alkaline solutions in low concentrations. To obtain the hydrossoluble pigment, higher concentrations are suggested, and they could be associated to heating.

Teor de bixina em quatro variedades de Bixa orellana L. cultivadas na Paraíba / Bixin content in four varieties of Bixa orellana L. grown in Paraíba

Neste trabalho foi verificado o teor de Óleos, sólidos e bixina em sementes de quatro variedades de Bixa orellana L. cultivadas na Paraíba: "Casca verde", "Casca vermelha", Bico de calango" e "Grão preto". Os melhores resultados foram obti­dos com os tipos "Casca verde" e "Casca vermelha" que apresentaram um rendimento em bixina pura, cristalina, de 1,3 e 1,1 por cento, respectivamente.

In this work it was verified the content of oils, solids and bixin in seeds of four varieties of Bixa orellana L. cultivated in Paraíba: "Casca verde", "Casca vermelha", Bico de calango" and "Grão preto". The best results were obtained with the types "Casca verde" and "Casca vermelha" that presented an yield in pure, crystalline bixina, of 1,3 and 1,1 percent, respectively.