BR102020009691A2 - PROCESS FOR OBTAINING AND PURIFICATION OF HYALURONIC ACID PRESENT IN THE BEARDS OF PERU - Google Patents
PROCESS FOR OBTAINING AND PURIFICATION OF HYALURONIC ACID PRESENT IN THE BEARDS OF PERU Download PDFInfo
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 53
- 229920002674 hyaluronan Polymers 0.000 title claims abstract description 53
- 229960003160 hyaluronic acid Drugs 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000000746 purification Methods 0.000 title description 4
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- 208000015122 neurodegenerative disease Diseases 0.000 claims abstract description 3
- 210000001179 synovial fluid Anatomy 0.000 claims abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 229960001927 cetylpyridinium chloride Drugs 0.000 claims description 11
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 claims description 11
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- 210000001519 tissue Anatomy 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 6
- 230000029087 digestion Effects 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims 1
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- RNAMYOYQYRYFQY-UHFFFAOYSA-N 2-(4,4-difluoropiperidin-1-yl)-6-methoxy-n-(1-propan-2-ylpiperidin-4-yl)-7-(3-pyrrolidin-1-ylpropoxy)quinazolin-4-amine Chemical compound N1=C(N2CCC(F)(F)CC2)N=C2C=C(OCCCN3CCCC3)C(OC)=CC2=C1NC1CCN(C(C)C)CC1 RNAMYOYQYRYFQY-UHFFFAOYSA-N 0.000 description 1
- 241000124815 Barbus barbus Species 0.000 description 1
- JRMSLDWZFJZLAS-UHFFFAOYSA-M [7-(dimethylamino)-1,9-dimethylphenothiazin-3-ylidene]-dimethylazanium;chloride Chemical compound [Cl-].CC1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC(C)=C3N=C21 JRMSLDWZFJZLAS-UHFFFAOYSA-M 0.000 description 1
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- HWEXKRHYVOGVDA-UHFFFAOYSA-M sodium;3-trimethylsilylpropane-1-sulfonate Chemical compound [Na+].C[Si](C)(C)CCCS([O-])(=O)=O HWEXKRHYVOGVDA-UHFFFAOYSA-M 0.000 description 1
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- Polysaccharides And Polysaccharide Derivatives (AREA)
- Cosmetics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
processo para obtenção e purificação do ácido hialurônico presente nas barbelas de peru. a presente invenção refere-se à descoberta de uma nova matriz para extração de ácido hialurônico da barbela de peru. a barbela está localizada abaixo do pescoço do peru é um subproduto de baixo valor comercial, proveniente da industrialização de peru (meleagris galopavos). estudos explorando subprodutos da industrialização de peru para fins mais nobres, ainda são escassos. sendo o brasil um dos principais produtores mundiais e a crescente produção nacional e elevada geração de sobras de processamento, esta invenção tem como objetivo valorizar comercialmente a barbela de peru, obtendo e purificando o ácido hialurônico presente nela. o ácido hialurônico é um produto de alto valor agregado que é obtido principalmente de cordão umbilical de mamíferos e da crista de galo, com uso garantido nas áreas médico-farmacêuticas e cosmética. são diversos os tratamentos onde o ácido hialurônico é utilizado, na área cosmética como meio hidratante e como meio para encapsulação e liberação controlada de fármacos e cosméticos, no tratamento de doenças degenerativas e inflamatórias das articulações dos ossos, na reposição de liquido sinovial, na liberação de agentes quimioterápicos em implantes cirúrgicos. o ácido hialurônico não é produzido comercialmente no brasil e a barbela de peru têm potencial para substituir a crista de galo que hoje é uma das principais fontes de obtenção de ácido hialurônico.process for obtaining and purifying the hyaluronic acid present in turkey dewlap. The present invention relates to the discovery of a new matrix for extracting hyaluronic acid from turkey dewlap. The dewlap is located below the turkey's neck and is a by-product of low commercial value, originating from the industrialization of turkeys (meleagris galopavos). Studies exploring byproducts of turkey industrialization for more noble purposes are still scarce. With Brazil being one of the world's main producers and the growing national production and high generation of processing leftovers, this invention aims to commercially enhance turkey barb, obtaining and purifying the hyaluronic acid present in it. Hyaluronic acid is a product with high added value that is obtained mainly from mammalian umbilical cord and rooster comb, with guaranteed use in the medical-pharmaceutical and cosmetic areas. There are several treatments where hyaluronic acid is used, in the cosmetic area as a moisturizing agent and as a means for encapsulation and controlled release of drugs and cosmetics, in the treatment of degenerative and inflammatory diseases of bone joints, in the replacement of synovial fluid, in the release of chemotherapy agents in surgical implants. Hyaluronic acid is not commercially produced in Brazil and turkey wattles have the potential to replace rooster combs, which today are one of the main sources of hyaluronic acid.
Description
[001] A presente patente de invenção refere-se a descoberta de um processo de obtenção de ácido hialurônico das barbelas que são um subproduto proveniente da industrialização de peru (Meleagris galopavos), que hoje é considerado um rejeito industrial, sem valor econômico, no entanto amplamente disponível no Brasil principalmente na região Sul.[001] The present invention patent refers to the discovery of a process for obtaining hyaluronic acid from barbs, which are a by-product from the industrialization of turkey (Meleagris galopavos), which today is considered an industrial waste, with no economic value, in the However widely available in Brazil, mainly in the South region.
[002] O Brasil não tem produção comercial de ácido hialurônico, mas hoje responde pela produção de 390,48 mil Ton/ano de carne de peru, sendo que cada indústria de abate de peru gera em média 5,931 Kg/mês de subprodutos, estes têm como principal destino a graxaria, sendo subutilizados para a fabricação de sabão, óleos e ração, produtos de baixo valor comercial.[002] Brazil does not have commercial production of hyaluronic acid, but today it accounts for the production of 390,48 thousand ton/year of turkey meat, with each turkey slaughter industry generating an average of 5,931 kg/month of by-products. their main destination is grease, being underused for the manufacture of soap, oils and animal feed, products with low commercial value.
[003] O ácido hialurônico faz parte da família dos glicosaminoglicanos, carboidratos de cadeia longa (polissacarídeos lineares), podendo ser obtido em maior quantidade em cordão umbilical e crista de galo, no entanto existente em outras fontes como o tecidos conjuntivos de animais, como pele e cartilagem, preenchendo os espaços intercelulares, com a importante função de flexibilidade e manutenção da estrutura dos tecidos.[003] Hyaluronic acid is part of the family of glycosaminoglycans, long-chain carbohydrates (linear polysaccharides), which can be obtained in greater quantity in umbilical cord and rooster comb, however existing in other sources such as connective tissues of animals, such as skin and cartilage, filling the intercellular spaces, with the important function of flexibility and maintenance of tissue structure.
[004] O ácido hialurônico possui alto valor agregado US$ 65,00/ 100mL, e este fato está correlacionado com o laborioso processo de obtenção, que se iniciou com a retirada do material lipídico e digestão utilizando o tampão de acetato de sódio em pH 5 e a enzima papaína á 60 °C, seguindo de etapas de precipitação com Cloreto de cetilpiridinio, centrifugação, liofilização e por fim uma separação e purificação do ácido hialurônico por coluna de cromatografia de troca iônica com resina DEAE-Sepharose Fast Flow.[004] Hyaluronic acid has a high added value of US$ 65.00/100mL, and this fact is correlated with the laborious process of obtaining it, which began with the removal of the lipid material and digestion using sodium acetate buffer at pH 5 and the enzyme papain at 60 °C, followed by precipitation steps with cetylpyridinium chloride, centrifugation, lyophilization and finally a separation and purification of hyaluronic acid by ion exchange chromatography column with DEAE-Sepharose Fast Flow resin.
[005] Nas áreas farmacêutica, médica e cosmética, o ácido hialurônico é versátil nas formas de hidrogel, andaimes, filmes, cremes, espumas e géis, e tem mostrado amplas variações de atividades farmacológicas, inclusive antidiabéticas, anti-envelhecimento e reparo da pele, antiproliferativo, anticâncer, antitumoral, anti-inflamatório, cicatrização de feridas e reparo tecidual.[005] In the pharmaceutical, medical and cosmetic areas, hyaluronic acid is versatile in hydrogel, scaffolding, films, creams, foams and gels, and has shown wide variations in pharmacological activities, including antidiabetic, anti-aging and skin repair , antiproliferative, anticancer, antitumor, anti-inflammatory, wound healing and tissue repair.
[006] Na base de patentes do Instituto Nacional de Propriedade Intelectual (INPI) documentos referentes á diferentes formas de obtenção de ácido hialurônico são publicados. Os documentos de BR 10 2017 027243 5 A2, PI 0713215-8 A2, PI 9205240- 1 A2, descrevem variados processos para a obtenção de ácido hialurônico. [006] In the patent base of the National Institute of Intellectual Property (INPI) documents referring to different ways of obtaining hyaluronic acid are published. The documents of BR 10 2017 027243 5 A2, PI 0713215-8 A2, PI 9205240-1 A2, describe various processes for obtaining hyaluronic acid.
[007] Assim como os pedidos PI 1000460-2 B1 se refere a extração de ácido hialurônico da crista do galo, por um método apenas químico, sem o uso de enzimas. [007] As well as PI 1000460-2 B1 requests, it refers to the extraction of hyaluronic acid from the comb of the rooster, by a chemical only method, without the use of enzymes.
[008] O documento WO2004080388A2, se refere á uma nova matriz a membrana da casca de ovo que foi utilizada para extração de ácido hialurônico. [008] The document WO2004080388A2 refers to a new eggshell membrane matrix that was used for the extraction of hyaluronic acid.
[009] Na base de patentes ESPACENET, á patentes existentes buscando aumentar o rendimento para obter mais ácido hialurônico, os documentos KR101443429B1, JOH02234689A, WO2011114472A1, CN106589164A, descrevem mudanças na metodologia para aumentar a quantidade de ácido hialurônico obtida.[009] Based on ESPACENET patents, existing patents seeking to increase the yield to obtain more hyaluronic acid, documents KR101443429B1, JOH02234689A, WO2011114472A1, CN106589164A, describe changes in the methodology to increase the amount of hyaluronic acid obtained.
[010] Os pedidos KR20030090501A, JPS63129991A referem-se á novos processos para melhorar a qualidade do ácido hialurônico e obter um composto com alta massa molecular.[010] Applications KR20030090501A, JPS63129991A refer to new processes to improve the quality of hyaluronic acid and obtain a compound with high molecular mass.
[011] Os documentos WO8906243A1, JP2009256463A, JP2010053259A, JP2631469B2, WO2004016771A1, referem-se á novos processos de purificação do ácido hialurônico.[011] WO8906243A1, JP2009256463A, JP2010053259A, JP2631469B2, WO2004016771A1, refer to new processes for the purification of hyaluronic acid.
[012] Na base de patentes PATENTSCOPE, o documento PI0707763 encontrado na base também se refere á um novo processo para obtenção de ácido hialurônico de alto massa molar.[012] On the basis of patents PATENTSCOPE, document PI0707763 found in the base also refers to a new process for obtaining hyaluronic acid of high molar mass.
[013] Nas bases EPOQUE, STN, USPTO não foram encontradas nenhuma patente relacionada obtenção de ácido hialurônico. [013] In the EPOQUE, STN, USPTO databases no patents related to obtaining hyaluronic acid were found.
[014] O presente pedido de patente refere-se ao processo de obtenção e purificação do ácido hialurônico obtido a partir de barbelas de peru (Meleagris galopavos), utilizando a papaína como enzima a temperatura de 60 °C em tampão de Acetato de sódio á pH 5,0. [014] The present patent application refers to the process of obtaining and purifying hyaluronic acid obtained from turkey barbs (Meleagris galopavos), using papain as an enzyme at a temperature of 60 °C in a sodium acetate buffer. pH 5.0.
[015] Procedimento 1 para extração de ácido hialurônico, etapa de retirada de resíduos lipídicos (deslipidação): Foi realizada seguindo a metodologia de Cardoso (1992) com modificações. As barbelas foram cortadas e a deslipidação ocorreu com imersão em acetona por 24 h á 4 °C, após foram realizadas 2 substituições de 24 horas cada com clorofórmio/metanol 24h a 25 °C. [015] Procedure 1 for extraction of hyaluronic acid, lipid residue removal step (delipidation): It was performed following the methodology of Cardoso (1992) with modifications. The dewlaps were cut and delipidated with immersion in acetone for 24 h at 4 °C, after which 2 replacements of 24 h each with chloroform/methanol 24 h at 25 °C were performed.
[016] Procedimento 2 para extração de ácido hialurônico, etapa de extração: Os tecidos secos foram hidratados em tampão de digestão Acetato de sódio pH 5,0 e hidratado com solução de papaína e incubados por 24 h a 60 °C, centrifugados e adicionado ao sobrenadante cloreto de cetilpiridínio (CPC) por 72 h a 8 °C ao sobrenadante, numa proporção de 0,2 mL para 100 mg de tecido seco e deixado em repouso por 72 h a 8 °C, a amostra seguiu para centrifugação o sobrenadante foi descartado; e o material precipitado o pellet foi lavado com 3,0 mL de solução de NaCl 2,0 M e etanol absoluto (100:15, v/v), posteriormente este sobrenadante resultante desta lavagem foi utilizado para uma segunda precipitação com 5% de CPC, na mesma proporção descrita anteriormente e deixado em repouso por 72 h a 8 °C;[016] Procedure 2 for extraction of hyaluronic acid, extraction step: Dried tissues were hydrated in digestion buffer Sodium acetate pH 5.0 and hydrated with papain solution and incubated for 24 h at 60 °C, centrifuged and added to supernatant cetylpyridinium chloride (CPC) for 72 h at 8 °C to the supernatant, in a proportion of 0.2 mL for 100 mg of dry tissue and left to rest for 72 h at 8 °C, the sample was continued for centrifugation, the supernatant was discarded; and the precipitated material, the pellet was washed with 3.0 mL of 2.0 M NaCl solution and absolute ethanol (100:15, v/v), then this supernatant resulting from this washing was used for a second precipitation with 5% of CPC, in the same proportion as described above and left to rest for 72 h at 8 °C;
[017] A amostra seguiu para centrifugação, o sobrenadante foi descartado e o pellet foi lavado com solução de NaCl 2,0 M e etanol absoluto. Na amostra resultante foram adicionados dois volumes de etanol absoluto, depois foi deixada em repouso por 24 h a 16°C. O pellet resultante do processo, congelado á -18 °C e liofilizado, que posteriormente foi purificado.[017] The sample followed for centrifugation, the supernatant was discarded and the pellet was washed with a 2.0 M NaCl solution and absolute ethanol. Two volumes of absolute ethanol were added to the resulting sample, then left to stand for 24 h at 16°C. The resulting pellet from the process, frozen at -18 °C and lyophilized, which was later purified.
[018] Procedimento 3 para extração de ácido hialurônico, etapa de separação e purificação: O produto obtido passou por uma etapa de fracionamento, uma separação dos compostos que ocorreu em uma coluna de cromatografia de troca iônica em coluna utilizando a resina DEAE-Sepharose Fast Flow equilibrada e eluída, também com mesmo tampão, até a completa remoção dos polissacarídeos não retidos. A amostra foi retidos na coluna e eluídos utilizando um gradiente de NaCl 0,25M, 0,50M, 0,75M, 1,0M, 2,0M e 4,0M em tampão acetato de sódio pH 5,0. As frações obtidas foram monitoradas através da reação metacromática produzida na presença de azul de 1,9 dimetilmetileno (DMB) (FARNDALE; SAYERS; BARRETT, 1982) e conteúdo de ácido urônico pelo método de carbazol, segundo Dische, (1946), posteriormente as frações foram dialisadas e liofilizadas.[018] Procedure 3 for hyaluronic acid extraction, separation and purification step: The product obtained went through a fractionation step, a separation of the compounds that took place in an ion exchange column chromatography column using DEAE-Sepharose Fast resin Balanced and eluted flow, also with the same buffer, until complete removal of unretained polysaccharides. The sample was retained on the column and eluted using a gradient of 0.25M, 0.50M, 0.75M, 1.0M, 2.0M and 4.0M NaCl in sodium acetate buffer pH 5.0. The fractions obtained were monitored through the metachromatic reaction produced in the presence of 1,9 dimethylmethylene blue (DMB) (FARNDALE; SAYERS; BARRETT, 1982) and uronic acid content by the carbazole method, according to Dische, (1946), later the fractions were dialyzed and lyophilized.
[019] Procedimento 4 para caracterização de ácido hialurônico, identificação e quantificação: O ácido hialurônico foi identificado por espectros de RMN de 13C em espectrômetro BRUKER modelo DPX-400SF. Para aquisição do espectro de RMN de 13C foi utilizado o solvente deuterado D2O, tendo como referência interna para δ = 0 ppm 4,4dimetil-4silapentano-1-sulfonato de sódio. Os parâmetros experimentais do equipamento para aquisição do espectro do composto obtido, em geral foram: 400,13 MHZ para 1H e 100,23 para 13C; largura de pulso 90o , 8.0 μs (13C) temperatura de 50 °C. A concentração do ácido hialurônico das frações foi determinada por meio de método químico, utilizando-se o reagente colorimétrico Carbazol (DISCHE, 1946). Esse método baseia-se no desenvolvimento de cor pela ação de compostos orgânicos como o reagente Carbazol. A leitura foi realizada no espectrofotômetro (Bel-photonics –UV- M-51) em comprimento de onda de A525 nm e a concentração do ácido hialurônico foi determinada por meio de uma curva de calibração previamente construída com o ácido hialurônico padrão (Sigma-Aldrich) tomado como referência. O ácido hialurônico da barbela do peru identificado é similar ao de crista de galo que foi tomado como referência. A concentração de ácido hialurônico encontrado na barbela de peru é de 12,0 μg de AH mg1 de tecido seco e a cristas de galo relatada por Rosa et al., (2008) 14,9μg de AH mg-1 de tecido seco.[019] Procedure 4 for characterization of hyaluronic acid, identification and quantification: Hyaluronic acid was identified by 13C NMR spectra in BRUKER model DPX-400SF spectrometer. For the acquisition of the 13C NMR spectrum, the deuterated solvent D2O was used, having as an internal reference δ = 0 ppm 4,4dimethyl-4silapentane-1-sulfonate sodium. The experimental parameters of the equipment for the acquisition of the spectrum of the obtained compound, in general, were: 400.13 MHZ for 1H and 100.23 for 13C; pulse width 90o , 8.0 μs (13C) temperature 50°C. The concentration of hyaluronic acid in the fractions was determined through a chemical method, using the colorimetric reagent Carbazol (DISCHE, 1946). This method is based on color development through the action of organic compounds such as Carbazol reagent. The reading was performed using a spectrophotometer (Bel-photonics –UV-M-51) at a wavelength of A525 nm and the concentration of hyaluronic acid was determined using a calibration curve previously constructed with standard hyaluronic acid (Sigma-Aldrich ) taken as a reference. The hyaluronic acid from the turkey dewlap identified is similar to the rooster's comb that was taken as a reference. The concentration of hyaluronic acid found in turkey barb is 12.0 μg of HA mg1 of dry tissue and the rooster combs reported by Rosa et al., (2008) 14.9 μg of HA mg-1 of dry tissue.
[020] O ácido hialurônico extraído das barbelas de peru é caracterizado por ser mais uma alternativa para extração deste composto que é amplamente utilizado pela indústria farmacêutica, no tratamento de doenças degenerativas e inflamatórias das articulações dos ossos, na reposição do liquido sinovial, na liberação de agentes quimioterápicos em implantes cirúrgicos e na área cosmética como meio hidratante. O ácido hialurônico têm potencial para ser utilizada na encapsulação e liberação controlada de fármacos por se tratar de um polissacarídeo atóxico pode ser utilizado pela indústria farmacêutica para o desenvolvimento de novos medicamentos e na indústria cosmética.[020] The hyaluronic acid extracted from turkey barbs is characterized by being another alternative for the extraction of this compound that is widely used by the pharmaceutical industry, in the treatment of degenerative and inflammatory diseases of the bone joints, in the replacement of synovial fluid, in the release of chemotherapeutic agents in surgical implants and in the cosmetic area as a moisturizing medium. Hyaluronic acid has the potential to be used in the encapsulation and controlled release of drugs, as it is a non-toxic polysaccharide, it can be used by the pharmaceutical industry for the development of new drugs and in the cosmetic industry.
[021] O ácido hialurônico é obtido atualmente de cordão umbilical e de crista de galo e é caracterizado pelo baixo rendimento e disponibilidade destas matérias primas, tendo em vista que dos frangos abatidos no Brasil nem todos são galos, á barbela vêm como uma alternativa a mais para obtenção do ácido hialurônico com larga disponibilidade, pela produção nacional de peru e que hoje é um rejeito industrial. [021] Hyaluronic acid is currently obtained from the umbilical cord and rooster's comb and is characterized by the low yield and availability of these raw materials, given that not all of the chickens slaughtered in Brazil are roosters, the barbel comes as an alternative to more to obtain widely available hyaluronic acid, through the national production of turkey, which today is an industrial waste.
[022] A invenção se refere, portanto, á uma nova matéria-prima as barbelas de peru como nova opção para obtenção de ácido hialurônico e valoração de subprodutos da industrialização de peru (Meleagris galopavos) um rejeito industrial de baixo valor comercial dentro de um mercado em expansão. [022] The invention therefore refers to a new raw material, turkey barbs as a new option for obtaining hyaluronic acid and valuation of by-products from the industrialization of turkey (Meleagris galopavos) an industrial waste of low commercial value within a expanding market.
[023] A seguir são apresentadas e descritas algumas figuras que ilustram o processo de obtenção e caracterização do ácido hialurônico. [023] Below are presented and described some figures that illustrate the process of obtaining and characterization of hyaluronic acid.
[024] Figura 1 – Apresenta o fluxograma do processo de extração de ácido hialurônico da barbela de peru (Meleagris galopavos). Neste fluxograma estão as diferentes operações unitárias realizadas para a obtenção do ácido hialurônico, as quais são: A extração dos glicosaminoglicanos (GAGs) totais foi realizada seguindo a metodologia de Cardoso et al., (1992) com modificações. [024] Figure 1 - Shows the flowchart of the process of extracting hyaluronic acid from turkey dewlap (Meleagris galopavos). In this flowchart are the different unit operations performed to obtain hyaluronic acid, which are: The extraction of total glycosaminoglycans (GAGs) was performed following the methodology of Cardoso et al., (1992) with modifications.
[025] Figura 2 – Apresenta o fracionamento dos glicosaminoglicanos totais das barbelas de peru foi realizado com o pellet de glicosaminoglicanos totais obtidos na extração. Este foi dissolvido no tampão e submetido à cromatografia de troca iônica em coluna de DEAE-Sepharose Fast Flow equilibrada e eluída, também com mesmo tampão, até a completa remoção dos polissacarídeos não retidos. Os glicosaminoglicanos retidos na coluna foram eluídos utilizando um gradiente de NaCl 0,25M, 0,50M, 0,75M, 1,0M, 2,0M e 4,0M.[025] Figure 2 - Shows the fractionation of total glycosaminoglycans of turkey barbs was performed with the pellet of total glycosaminoglycans obtained in the extraction. This was dissolved in the buffer and submitted to ion exchange chromatography on a balanced and eluted DEAE-Sepharose Fast Flow column, also with the same buffer, until the complete removal of non-retained polysaccharides. The glycosaminoglycans retained on the column were eluted using a gradient of 0.25M, 0.50M, 0.75M, 1.0M, 2.0M and 4.0M NaCl.
[026] Figura 3 – Apresenta o espectro de RMN1H do ácido hialurônico extraído da barbela de peru (a) comparado ao obtido de ácido hialurônico padrão de crista de galo da Sigma-Aldrich (b), em D2O. Os dados apresentados pelo RMN de 1H para o ácido hialurônico de barbela de peru são similares aos obtidos para ácido hialurônico padrão, bem como com os descritos na literatura (YANG et al., 2011; REDDY; KARUNAKARAN, 2013; WU et al., 2013) comprovando a identificação da estrutura como sendo o ácido hialurônico. [026] Figure 3 – Shows the 1H NMR spectrum of hyaluronic acid extracted from turkey barb (a) compared to that obtained from standard hyaluronic acid from Sigma-Aldrich rooster comb (b), in D2O. The data presented by 1H NMR for turkey barb hyaluronic acid are similar to those obtained for standard hyaluronic acid, as well as those described in the literature (YANG et al., 2011; REDDY; KARUNAKARAN, 2013; WU et al., 2013) confirming the identification of the structure as being hyaluronic acid.
Claims (9)
- a. Retirada do material lipídico e secagem;
- b. Hidratação dos tecidos secos com tampão de digestão;
- c. Incubação com a enzima papaína;
- d. Precipitações do ácido hialurônico com cloreto de cetilpiridinio (CPC);
- e. Retirada do cloreto de cetilpiridinio;
- f. Cromatografia de troca iônica com resina DEAE- Sepharose Fast Flow;
- The. Removal of lipid material and drying;
- B. Hydration of dry tissues with digestion buffer;
- ç. Incubation with papain enzyme;
- d. Precipitation of hyaluronic acid with cetylpyridinium chloride (CPC);
- and. Removal of cetylpyridinium chloride;
- f. Ion exchange chromatography with DEAE-Sepharose Fast Flow resin;
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