BR102018069314A2 - BACCHARIS DRACUNCULIFOLIA D.C. ESSENTIAL OIL PURIFICATION PROCESS - Google Patents
BACCHARIS DRACUNCULIFOLIA D.C. ESSENTIAL OIL PURIFICATION PROCESS Download PDFInfo
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- LCWMKIHBLJLORW-UHFFFAOYSA-N gamma-carene Natural products C1CC(=C)CC2C(C)(C)C21 LCWMKIHBLJLORW-UHFFFAOYSA-N 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- OJIGFVZZEVQUNV-UHFFFAOYSA-N germacrene D Natural products CC(C)C1CCC=C(/C)CCC(=C)C=C1 OJIGFVZZEVQUNV-UHFFFAOYSA-N 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- GAIBLDCXCZKKJE-UHFFFAOYSA-N isogermacrene D Natural products CC(C)C1CCC(C)=CCCC(=C)C=C1 GAIBLDCXCZKKJE-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000001510 limonene Nutrition 0.000 description 1
- 229940087305 limonene Drugs 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229960001019 oxacillin Drugs 0.000 description 1
- UWYHMGVUTGAWSP-JKIFEVAISA-N oxacillin Chemical compound N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C1=C(C)ON=C1C1=CC=CC=C1 UWYHMGVUTGAWSP-JKIFEVAISA-N 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229940069949 propolis Drugs 0.000 description 1
- GRWFGVWFFZKLTI-UHFFFAOYSA-N rac-alpha-Pinene Natural products CC1=CCC2C(C)(C)C1C2 GRWFGVWFFZKLTI-UHFFFAOYSA-N 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000002940 repellent Effects 0.000 description 1
- 239000005871 repellent Substances 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 229930004725 sesquiterpene Natural products 0.000 description 1
- 150000004354 sesquiterpene derivatives Chemical class 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- USDOQCCMRDNVAH-UHFFFAOYSA-N sigma-cadinene Natural products C1C=C(C)CC2C(C(C)C)CC=C(C)C21 USDOQCCMRDNVAH-UHFFFAOYSA-N 0.000 description 1
- 239000000344 soap Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- AYXPYQRXGNDJFU-IMNVLQEYSA-N viridiflorol Chemical compound [C@@H]1([C@@](CC[C@@H]2[C@H]3C2(C)C)(C)O)[C@H]3[C@H](C)CC1 AYXPYQRXGNDJFU-IMNVLQEYSA-N 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- IHPKGUQCSIINRJ-UHFFFAOYSA-N β-ocimene Natural products CC(C)=CCC=C(C)C=C IHPKGUQCSIINRJ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
a presente invenção refere-se a um processo de purificação do óleo essencial de baccharis dracunculifolia d.c. para obtenção de frações contendo alto teor de trans-nerolidol (de 91,47% a 96,14%) e espatulenol (de 78,63 a 84,89%).the present invention relates to a process for purifying the essential oil of baccharis dracunculifolia d.c. to obtain fractions containing a high content of trans-nerolidol (from 91.47% to 96.14%) and spatulenol (from 78.63 to 84.89%).
Description
PROCESSO DE PURIFICAÇÃO DO ÓLEO ESSENCIAL DE BACCHARIS DRACUNCULIFOLIA D.C.BACCHARIS DRACUNCULIFOLIA D.C. ESSENTIAL OIL PURIFICATION PROCESS
Campo da invenção:Field of the invention:
[001] A presente invenção se insere no campo de aplicação da química de óleos vegetais, mais especificamente na área de recuperação ou refinação de óleos essenciais a partir de matérias-primas, uma vez que se refere a um processo de purificação do óleo essencial de Baccharis dracunculifolia D.C. (Asteraceae) para obtenção de frações contendo trans-nerolidol ((6E)-3,7,11-Trimethyl-1,6,10dodecatrien-3-ol) e espatulenol ((1aR,4aR,7S,7aR,7bR)1,1,7-Trimethyl-4-methylenedecahydro-1Hcyclopropa[e]azulen-7-ol).[001] The present invention falls within the field of application of vegetable oil chemistry, more specifically in the area of recovery or refining essential oils from raw materials, since it refers to a process of purification of essential oil from Baccharis dracunculifolia DC (Asteraceae) for obtaining fractions containing trans-nerolidol ((6E) -3,7,11-Trimethyl-1,6,10dodecatrien-3-ol) and spatulenol ((1aR, 4aR, 7S, 7aR, 7bR ) 1,1,7-Trimethyl-4-methylenedecahydro-1Hcyclopropa [e] azulen-7-ol).
Fundamentos da invenção:Basics of the invention:
[002] Baccharis dracunculifolia D.C. (Asteraceae) é uma planta nativa do Brasil que vem sendo utilizada na medicina popular como antipirético, anti-inflamatório, antisséptico e no tratamento de feridas cutâneas e desordens gastrointestinais. Essa planta, também conhecida como alecrim do campo, vassoura ou vassourinha, é a principal fonte botânica da própolis verde.[002] Baccharis dracunculifolia D.C. (Asteraceae) is a plant native to Brazil that has been used in folk medicine as antipyretic, anti-inflammatory, antiseptic and in the treatment of skin wounds and gastrointestinal disorders. This plant, also known as field rosemary, broom or broom, is the main botanical source of green propolis.
[003] Com um aroma forte e exótico, o óleo essencial obtido das folhas da planta Baccharis dracunculifolia é uma importante matéria-prima para uso em cosméticos e em perfumes. Além disso, o óleo essencial apresenta diversas atividades biológicas com ação antimicrobiana, antifúngica, acaricida, contra úlcera gástrica, entre outros. Dentre os principais constituintes do óleo essencial de Baccharis dracunculifolia, destacam-se, os sesquiterpenos transnerolidol e o espatulenol, que têm alto valor comercial.[003] With a strong and exotic aroma, the essential oil obtained from the leaves of the Baccharis dracunculifolia plant is an important raw material for use in cosmetics and perfumes. In addition, the essential oil has several biological activities with antimicrobial, antifungal, acaricidal action, against gastric ulcer, among others. Among the main constituents of the essential oil of Baccharis dracunculifolia, the most important are the sesquiterpenes transnerolidol and espatulenol, which have high commercial value.
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2/24 [004] O nerolidol é um composto que apresenta uma fragrância floral e está presente em óleos essenciais de diversas plantas. Com um consumo anual de 10 a 100 toneladas, esta substância é amplamente utilizada no preparo de fragrâncias, shampoos, sabonetes e detergentes, e também é utilizada na indústria de alimentos, sendo aprovada pelo U.S. Food and Drug Administration (FDA) para uso como aromatizantes. Alguns estudos sugerem que o composto espatulenol apresenta atividade repelente, atividade imunomodulatória, anti-inflamatória e, ainda, que possa ser um bom candidato para uso no tratamento de câncer. Aplicações abrangidas por patentes incluem o uso deste composto em perfumes, fragrâncias, cosméticos, detergentes e alimentos.2/24 [004] Nerolidol is a compound that has a floral fragrance and is present in essential oils from different plants. With an annual consumption of 10 to 100 tons, this substance is widely used in the preparation of fragrances, shampoos, soaps and detergents, and is also used in the food industry, being approved by the US Food and Drug Administration (FDA) for use as flavorings. . Some studies suggest that the compound spatulenol has repellent activity, immunomodulatory, anti-inflammatory activity and, even, that it may be a good candidate for use in the treatment of cancer. Applications covered by patents include the use of this compound in perfumes, fragrances, cosmetics, detergents and foods.
[005] Nerolidol e espatulenol têm diversas propriedades biológicas conhecidas, com um potencial farmacológico e terapêutico de grande interesse para a indústria farmacêutica. Contudo, a obtenção desses compostos através de síntese química pode apresentar alto custo e produção com baixo rendimento. Assim, o desenvolvimento de métodos rápidos, de baixo custo e com maior rendimento é essencial para a produção destes compostos em grande escala.[005] Nerolidol and espatulenol have several known biological properties, with pharmacological and therapeutic potential of great interest to the pharmaceutical industry. However, obtaining these compounds through chemical synthesis can have high cost and low yield production. Thus, the development of fast, low cost and higher yield methods is essential for the production of these compounds on a large scale.
[006] Nesse sentido, a presente invenção propõe um processo de purificação do óleo essencial da planta B. dracunculifolia D.C., em que a extração do óleo é realizada através do processo de hidrodestilação a partir de folhas e ramos frescos da referida planta, com posterior realização de cromatografia Flash (cromatografia à média pressão) resultando em frações contendo alto teor dos compostos transnerolidol e espatulenol. Além disso, direciona o fracionamento para o isolamento dos compostos trans[006] In this sense, the present invention proposes a process for purifying the essential oil of the plant B. dracunculifolia DC, in which the oil extraction is carried out through the process of hydrodistillation from fresh leaves and branches of the referred plant, with subsequent Flash chromatography (medium pressure chromatography) resulting in fractions containing a high content of transnerolidol and spatulenol compounds. In addition, it directs fractionation to isolate trans compounds
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3/24 nerolidol e espatulenol utilizando um equipamento detector de U.V. com seleção de comprimentos de onda específicos para coletar frações contendo estes compostos. Assim, o presente processo permite uma alternativa rápida e de baixo custo para obtenção de frações com alto teor de trans-nerolidol (de 91,47% a 96,14%) e espatulenol (de 78,63 a 84,89%).3/24 nerolidol and spatulenol using U.V. with selection of specific wavelengths to collect fractions containing these compounds. Thus, the present process allows a fast and low cost alternative to obtain fractions with a high content of trans-nerolidol (from 91.47% to 96.14%) and spatulenol (from 78.63 to 84.89%).
[007] Alguns documentos do estado da técnica descrevem processos de extração de óleos essenciais.[007] Some state-of-the-art documents describe processes for extracting essential oils.
[008] O artigo “Chemical composition of the essential oils of Baccharis species from southern Brazil: a comparative study using multivariate statistical analysis” de Souza et. al. (2017) descreve um processo para obtenção de óleos essenciais e um método de análise química de óleos essenciais de diferentes espécies de Baccharis. Todavia, além de não utilizar a espécie alvo Baccharis dracunculifolia D.C., o processo revelado no referido artigo se distancia da presente invenção por apresentar etapas e parâmetros distintos, tais como etapas adicionais de extração, ausência de fracionamento do óleo, ausência de purificação e, principalmente, não obter frações enriquecidas em espatulenol e trans-nerolidol.[008] The article “Chemical composition of the essential oils of Baccharis species from southern Brazil: a comparative study using multivariate statistical analysis” by Souza et. al. (2017) describes a process for obtaining essential oils and a method of chemical analysis of essential oils of different species of Baccharis. However, in addition to not using the target species Baccharis dracunculifolia DC, the process revealed in that article differs from the present invention as it presents different stages and parameters, such as additional extraction steps, absence of oil fractionation, absence of purification and, mainly , do not obtain fractions enriched in spatulenol and trans-nerolidol.
[009] O documento BRPI0800363-7 A2 descreve um processo de extração e fracionamento dos extratos brutos de espécies de Baccharis, para obtenção de frações ricas em seus princípios ativos, por cromatografia por gravidade em colunas empacotadas com sílica gel, de partição líquidolíquido (utilizando somente solventes para a separação), de fracionamento por extração ácido-base e/ou de utilização de coluna líquida contendo sílica gel e eluição com solventes orgânicos de diferentes polaridades. Diferentemente, a[009] The document BRPI0800363-7 A2 describes a process of extraction and fractionation of crude extracts of Baccharis species, to obtain fractions rich in their active principles, by gravity chromatography in columns packed with silica gel, of liquid partition (using liquid) solvents for separation only), fractionation by acid-base extraction and / or using a liquid column containing silica gel and elution with organic solvents of different polarities. In contrast, the
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4/24 presente invenção propõe a realização de cromatografia Flash (cromatografia à média pressão) para o fracionamento do óleo essencial, utilizando cartucho com partículas de sílica como fase estacionária e solventes orgânicos ou mistura de solventes como fase móvel, além do uso do detector de luz ultravioleta (U.V.) utilizado para a coleta das frações contendo os compostos de interesse (trans-nerolidol e espatulenol). Além disso, os compostos majoritários presentes nas frações de óleo essencial do presente invento (trans-nerolidol e espatulenol) diferem dos compostos obtidos no extrato de Baccharis dracuncullfolla relatados no documento em questão.The present invention proposes the performance of Flash chromatography (medium pressure chromatography) for the fractionation of essential oil, using a cartridge with silica particles as a stationary phase and organic solvents or mixture of solvents as a mobile phase, in addition to the use of the ultraviolet (UV) light used to collect fractions containing the compounds of interest (trans-nerolidol and spatulenol). In addition, the major compounds present in the essential oil fractions of the present invention (trans-nerolidol and spatulenol) differ from the compounds obtained in the Baccharis dracuncullfolla extract reported in the document in question.
[010] Já os documentos WO16092376 A1 e WO0072861 A1 descrevem um processo de extração e purificação de substâncias ativas de várias plantas, incluindo o isolamento de compostos terpenóides. Contudo, diferentemente da presente invenção que obtém os compostos-alvo espatulenol e trans-nerolidol do óleo essencial, os referidos documentos descrevem a obtenção de um extrato, e por esse motivo, as etapas são distantes das etapas aqui propostas, e ainda não detalham condições para obtenção de frações enriquecidas nos compostos-alvo da presente invenção.[010] Documents WO16092376 A1 and WO0072861 A1 describe a process for the extraction and purification of active substances from various plants, including the isolation of terpenoid compounds. However, unlike the present invention which obtains the target compounds spatulenol and trans-nerolidol from the essential oil, these documents describe obtaining an extract, and for this reason, the steps are far from the steps proposed here, and do not yet detail conditions to obtain fractions enriched in the target compounds of the present invention.
[011] Portanto, nenhum documento do estado da técnica descreve um processo de purificação do óleo essencial da planta B. dracuncullfolla D.C. para obtenção de frações com alto teor de trans-nerolidol (de 91,47% a 96,14%) e espatulenol (de 78,63 a 84,89%), tal como proposto pela presente invenção.[011] Therefore, no state-of-the-art document describes a process for purifying the essential oil of the B. dracuncullfolla DC plant to obtain fractions with a high content of trans-nerolidol (from 91.47% to 96.14%) and spatulenol (from 78.63 to 84.89%), as proposed by the present invention.
Breve descrição da invenção:Brief description of the invention:
[012] A presente invenção refere-se a um processo[012] The present invention relates to a process
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5/24 de purificação do óleo essencial de Baccharis dracunculifolia D.C. (Asteraceae) para obtenção de frações contendo alto teor de trans-nerolidol (de 91,47% a 96,14%) e espatulenol (de 78,63 a 84,89%). O mesmo compreende as etapas de: a) extração do óleo essencial (OE) por hidrodestilação de partes aéreas da planta Baccharis dracunculifolia D.C.; b) fracionamento por cromatografia à média pressão (cromatografia Flash) com cartucho com partículas de sílica como fase estacionária, e solventes orgânicos ou mistura de solventes como fase móvel; c) realização de outro fracionamento por cromatografia à média pressão (cromatografia Flash), de frações obtidas na etapa anterior, com cartucho com partículas de sílica como fase estacionária, e solventes orgânicos ou mistura de solventes como fase móvel; d) eliminação dos solventes por fluxo de nitrogênio para posterior quantificação dos compostos-alvos; e) realização da identificação dos compostos presentes nas frações por cromatografia gasosa acoplada à espectrometria de massas (GC-MS); e f) realização da quantificação dos compostos presentes nas frações por cromatografia gasosa com detector por ionização em chama (GC-FID).5/24 purification of the essential oil of Baccharis dracunculifolia DC (Asteraceae) to obtain fractions containing a high content of trans-nerolidol (from 91.47% to 96.14%) and spatulenol (from 78.63 to 84.89% ). It comprises the steps of: a) extraction of essential oil (OE) by hydrodistillation from aerial parts of the Baccharis dracunculifolia D.C plant; b) fractionation by medium pressure chromatography (Flash chromatography) with a cartridge with silica particles as a stationary phase, and organic solvents or mixture of solvents as a mobile phase; c) performing another fractionation by medium pressure chromatography (Flash chromatography), of fractions obtained in the previous step, with a cartridge with silica particles as a stationary phase, and organic solvents or mixture of solvents as a mobile phase; d) elimination of solvents by nitrogen flow for subsequent quantification of target compounds; e) identification of the compounds present in the fractions by gas chromatography coupled to mass spectrometry (GC-MS); and f) quantification of the compounds present in the fractions by gas chromatography with flame ionization detector (GC-FID).
Breve descrição das figuras:Brief description of the figures:
[013] Para obter uma total e completa visualização do objeto desta invenção, são apresentadas as figuras as quais se faz referências, conforme se segue.[013] In order to obtain a total and complete visualization of the object of this invention, the figures to which reference is made are presented, as follows.
[014] A Figura 1 apresenta um fluxograma de uma exemplificação do processo de extração e fracionamento do óleo essencial de Baccharis dracunculifolia D.C. para obtenção de frações com alto teor de trans-nerolidol e espatulenol.[014] Figure 1 presents a flowchart of an example of the extraction and fractionation process of the essential oil of Baccharis dracunculifolia D.C. to obtain fractions with a high content of trans-nerolidol and spatulenol.
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6/246/24
cromatograma obtido no processo de purificação do óleo essencial de Baccharis dracunculifolia D.C. no equipamento Biotage IsoleraTM, utilizando um gradiente com os solventes n-Hexano (A) e acetato de etila (B).chromatogram obtained in the purification process of the essential oil of Baccharis dracunculifolia DC in the Biotage Isolera TM equipment, using a gradient with the solvents n-Hexane (A) and ethyl acetate (B).
[017] A Figura 4 apresenta graficamente o espectro de massas do trans-nerolidol obtido por GC/MS ion trap.[017] Figure 4 shows graphically the mass spectrum of trans-nerolidol obtained by GC / MS ion trap.
[018] A Figura 5 apresenta graficamente o espectro de massas do espatulenol obtido por GC-MS ion trap.[018] Figure 5 shows graphically the mass spectrum of spatulenol obtained by GC-MS ion trap.
Descrição detalhada da invenção:Detailed description of the invention:
[019] A presente invenção refere-se a um processo de purificação do óleo essencial de Baccharis dracunculifolia D.C. (Asteraceae) para obtenção de frações contendo altos teores de trans-nerolidol e espatulenol.[019] The present invention relates to a process of purifying the essential oil of Baccharis dracunculifolia D.C. (Asteraceae) to obtain fractions containing high levels of trans-nerolidol and spatulenol.
[020] O referido processo compreende as etapas de:[020] This process comprises the steps of:
a) Extração do óleo essencial (OE) por hidrodestilação de partes aéreas da planta Baccharis dracunculifolia D.C.;a) Extraction of essential oil (OE) by hydrodistillation of aerial parts of the plant Baccharis dracunculifolia D.C .;
b) Fracionamento por cromatografia à média pressão (cromatografia Flash) com cartucho com partículas de sílica como fase estacionária, e solventes orgânicos ou mistura de solventes como fase móvel;b) Fractionation by medium pressure chromatography (Flash chromatography) with a cartridge with silica particles as a stationary phase, and organic solvents or mixture of solvents as a mobile phase;
c) Realização de outro fracionamento por cromatografia à média pressão (cromatografia Flash) de frações obtidas na etapa anterior com cartucho com partículas de sílica como fase estacionária, e solventes orgânicos ou mistura de solventes como fase móvel;c) Performing another fractionation by medium pressure chromatography (Flash chromatography) of fractions obtained in the previous step with a cartridge with silica particles as a stationary phase, and organic solvents or mixture of solvents as a mobile phase;
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7/247/24
d) Eliminação dos solventes por fluxo de nitrogênio para posterior quantificação dos compostos-alvos;d) Elimination of solvents by nitrogen flow for subsequent quantification of target compounds;
e) Identificação dos compostos presentes nas frações por cromatografia gasosa acoplada à espectrometria de massas (GC-MS); ee) Identification of the compounds present in the fractions by gas chromatography coupled to mass spectrometry (GC-MS); and
f) Quantificação dos compostos presentes nas frações por cromatografia gasosa com detector por ionização em chama (GC-FID).f) Quantification of the compounds present in the fractions by gas chromatography with flame ionization detector (GC-FID).
[021] Na etapa “a” as partes aéreas são cortadas em pedaços médios que variam entre 5 a 10 cm, em que preferencialmente as partes aéreas são ramos e folhas frescas (in natura). É válido ressaltar que a utilização de ramos e folhas apresenta potencial reflexo na composição do óleo essencial que será obtido.[021] In step “a” the aerial parts are cut into medium pieces that vary between 5 to 10 cm, in which the aerial parts are preferably fresh branches and leaves (in natura). It is worth noting that the use of branches and leaves has a potential reflection on the composition of the essential oil that will be obtained.
[022] Ainda na etapa a, mais especificamente, utiliza-se de 0,01% a 0,10% (p/v) de pedaços de partes aéreas cortados, misturados em água e submetidos ao método de hidrodestilação (Li Y., Fabiano-Tixier AS., Chemat F.[022] Still in step a, more specifically, 0.01% to 0.10% (w / v) of pieces of cut aerial parts are used, mixed in water and subjected to the hydrodistillation method (Li Y., Fabiano-Tixier AS., Chemat F.
(2014) Essential Oils: From Conventional to Green Extraction. In: Essential Oils as Reagents in Green Chemistry. Springer Briefs in Molecular Science. Springer, Cham) por 3 a 4 horas utilizando aparato do tipo Clevenger, obtendo-se um rendimento de 0,22 a 0,37% de óleo essencial. Caso o processo não seja contínuo, o óleo essencial obtido pode ser armazenado em frascos âmbar em freezer à temperatura de -20 °C, para posterior uso na etapa b.(2014) Essential Oils: From Conventional to Green Extraction. In: Essential Oils as Reagents in Green Chemistry. Springer Briefs in Molecular Science. Springer, Cham) for 3 to 4 hours using Clevenger type apparatus, obtaining a yield of 0.22 to 0.37% of essential oil. If the process is not continuous, the essential oil obtained can be stored in amber bottles in a freezer at a temperature of -20 ° C, for later use in step b.
[023] Na etapa b, 0,05 g a 100 g do óleo essencial obtido na etapa anterior é fracionado por cromatografia à média pressão (cromatografia Flash) utilizando cartuchos de 50 a 1500 g empacotados com partículas de sílica que variam[023] In step b, 0.05 g to 100 g of the essential oil obtained in the previous step is fractionated by medium pressure chromatography (Flash chromatography) using cartridges of 50 to 1500 g packed with silica particles that vary
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8/24 de 30 a 70 pm como fase estacionária, e solventes orgânicos ou mistura de solventes como fase móvel selecionados do grupo que consiste em: metanol, acetonitrila, clorofórmio, diclorometano e ciclohexano, sendo preferencialmente os solventes n-hexano e acetato de etila, em um fluxo de 30 a 50 mL/minuto para cartuchos com 50 a 100 g e fluxo de 300 a 500 mL/minuto para cartuchos de 1500 g, por 10 a 60 minutos,8/24 from 30 to 70 pm as a stationary phase, and organic solvents or mixture of solvents as a mobile phase selected from the group consisting of: methanol, acetonitrile, chloroform, dichloromethane and cyclohexane, preferably being the solvents n-hexane and ethyl acetate , in a flow of 30 to 50 mL / minute for cartridges with 50 to 100 g and flow of 300 to 500 mL / minute for cartridges of 1500 g, for 10 to 60 minutes,
selecionados do grupo que consiste em:selected from the group consisting of:
- 100% de n-hexano por 0 a 3 minutos;- 100% n-hexane for 0 to 3 minutes;
- 100% a 65% de n-hexano e 0 a 35% de acetato de etila por 3 a 21 minutos.- 100% to 65% n-hexane and 0 to 35% ethyl acetate for 3 to 21 minutes.
[024] Sendo que à medida que se aumenta a proporção de acetato de etila, reduz-se a proporção de n-hexano [025] Mais preferencialmente, os solventes utilizados para a separação apresentam os gradientes selecionados do grupo que consiste em:[024] As the proportion of ethyl acetate increases, the proportion of n-hexane is reduced [025] More preferably, the solvents used for the separation show the gradients selected from the group consisting of:
por 18 a 21 minutos.for 18 to 21 minutes.
[026] Assim, através de um equipamento detector de luz ultravioleta (U.V.), as frações são coletadas conforme a detecção dos picos dos compostos-alvo (trans-nerolidol e[026] Thus, using ultraviolet light detector equipment (U.V.), the fractions are collected according to the detection of the peaks of the target compounds (trans-nerolidol and
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9/24 espatulenol) nos comprimentos de onda 254 nm e 280 nm.9/24 spatulenol) at 254 nm and 280 nm wavelengths.
[027] Na etapa c, é realizado outro fracionamento de frações obtidas nos cumprimentos de onda de 254 nm e 280 nm para a obtenção de uma fração mais pura do composto espatulenol. Para tal, 0,05 g a 100 g de frações são submetidas à cromatografia à média pressão (cromatografia Flash) utilizando cartucho de 50 a 1500 g empacotados com partículas de sílica que variam de 30 a 70 pm como fase estacionária, e solventes orgânicos ou mistura de solventes como fase móvel selecionados do grupo que consiste em: metanol, acetonitrila, clorofórmio, n-hexano e acetato de etila, sendo preferencialmente os solventes ciclohexano e diclorometano, um fluxo de 30 a 50 mL/minuto para cartuchos com 50 a 100 g e fluxo de 300 a 500 mL/min para cartuchos de 1500 g, por 10 a 60 minutos, e um volume máximo de frações coletadas por tubo de 10 a 20 mL para cartuchos de 50 a 100 g e de 240 a 480 mL para cartuchos de 1500g. Preferencialmente, os solventes utilizados para a separação apresentam os gradientes selecionados do grupo que consiste em:[027] In step c, another fractionation of fractions obtained in the wave lengths of 254 nm and 280 nm is carried out to obtain a purer fraction of the spatulenol compound. For this purpose, 0.05 g to 100 g of fractions are subjected to medium pressure chromatography (Flash chromatography) using a 50 to 1500 g cartridge packaged with silica particles ranging from 30 to 70 pm as a stationary phase, and organic solvents or mixture of solvents as a mobile phase selected from the group consisting of: methanol, acetonitrile, chloroform, n-hexane and ethyl acetate, preferably the solvents cyclohexane and dichloromethane, a flow of 30 to 50 mL / minute for cartridges with 50 to 100 g flow of 300 to 500 mL / min for cartridges of 1500 g, for 10 to 60 minutes, and a maximum volume of fractions collected per tube of 10 to 20 mL for cartridges of 50 to 100 g and of 240 to 480 mL for cartridges of 1500 g . Preferably, the solvents used for the separation present the gradients selected from the group consisting of:
- 40 a 0% de ciclohexano e 60 a 100% de diclorometano por 0 a 29 minutos.- 40 to 0% cyclohexane and 60 to 100% dichloromethane for 0 to 29 minutes.
[028] Sendo que à medida que se aumenta a proporção de diclorometano, reduz-se a proporção de ciclohexano [029] Mais preferencialmente, os solventes utilizados para a separação apresentam os gradientes selecionados do grupo que consiste em:[028] As the proportion of dichloromethane is increased, the proportion of cyclohexane is reduced [029] More preferably, the solvents used for the separation show the gradients selected from the group consisting of:
- 40% de ciclohexano e 60% de diclorometano por 0 a 4 minutos;- 40% cyclohexane and 60% dichloromethane for 0 to 4 minutes;
- 40% a 0% de ciclohexano e 60 a 100% de diclorometano- 40% to 0% cyclohexane and 60 to 100% dichloromethane
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10/24 por 4 a 18 minutos;10/24 for 4 to 18 minutes;
- 100% de diclorometano por 18 a 29 minutos.- 100% dichloromethane for 18 to 29 minutes.
[030] Assim, através de um equipamento detector de luz ultravioleta (U.V.), as frações são coletadas conforme a detecção do pico do composto-alvo nos comprimentos de onda 210 nm e 254nm.[030] Thus, using ultraviolet light detector equipment (U.V.), fractions are collected according to the detection of the peak of the target compound at wavelengths 210 nm and 254nm.
[031] Coletada as frações dos compostos-alvos, na etapa “d” é realizada a eliminação dos solventes nas referidas frações obtidas. O solvente pode ser eliminado por fluxo de nitrogênio, evaporador rotativo e concentrador a vácuo, na concretização da invenção, sem, no entanto, restringir a esta modalidade, é eliminado por fluxo de nitrogênio.[031] Once the fractions of the target compounds are collected, in step “d” the solvents are eliminated in the fractions obtained. The solvent can be eliminated by nitrogen flow, rotary evaporator and vacuum concentrator, in the embodiment of the invention, without, however, restricting to this modality, it is eliminated by nitrogen flow.
[032] Os solventes devem ser eliminados para injetar nos equipamentos CG-MS e CG-FID para que as amostras sejam diluídas em concentrações adequadas a fim de evitar o excesso ou falta de amostra, o que interfere nos cromatogramas.[032] Solvents must be eliminated to inject into the CG-MS and CG-FID equipment so that the samples are diluted in appropriate concentrations in order to avoid excess or lack of sample, which interferes with the chromatograms.
[033] Os compostos de interesse são obtidos ao final da etapa c. No entanto, dependendo da aplicação no que estes serão utilizados, como por exemplo, uso dos compostos em formulações, é necessário eliminar os solventes para preparar as soluções contendo os compostos nas concentrações desejadas e também para evitar inferências destes solventes na formulação.[033] The compounds of interest are obtained at the end of step c. However, depending on the application in which they will be used, for example, use of the compounds in formulations, it is necessary to eliminate the solvents to prepare the solutions containing the compounds in the desired concentrations and also to avoid inferences of these solvents in the formulation.
[034] Na etapa e é realizada a identificação dos compostos presentes nas frações obtidas. Para tal, em um cromatógrafo gasoso acoplado a espectrômetro de massas (CGMS), utiliza-se uma coluna com fase estacionária contendo 5% difenil e 95% dimetilpolisiloxano (por exemplo, OV-5ms, AT[034] In step e, the compounds present in the obtained fractions are identified. For this purpose, a gas chromatograph coupled to a mass spectrometer (CGMS) uses a column with a stationary phase containing 5% diphenyl and 95% dimethylpolysiloxane (for example, OV-5ms, AT
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11/2411/24
5ms, BP-5ms, HP-5ms, HP-5ms UI ou outra similar) (30 m x 0,25 mm x 0,25 pm), em que como gás de arraste utiliza-se hélio com fluxo 1 mL/min; volume de injeção de 1 pL; temperatura do injetor de 220 °C; e injeção realizada em modo splitless. A temperatura do forno é programada para 50 °C por 2 minutos, uma rampa de 50 °C a 310 °C aumentando 3 °C por minutos e espera a 310 °C por 20 minutos. O espectro de massas é gerado em modo Scan a 70 eV com faixa de massas de 41 a 450 m/z.5ms, BP-5ms, HP-5ms, HP-5ms UI or other similar) (30 m x 0.25 mm x 0.25 pm), in which helium gas is used with a flow rate of 1 mL / min; injection volume of 1 pL; injector temperature of 220 ° C; and injection performed in splitless mode. The oven temperature is programmed to 50 ° C for 2 minutes, a ramp from 50 ° C to 310 ° C increasing by 3 ° C for minutes and waiting at 310 ° C for 20 minutes. The mass spectrum is generated in Scan mode at 70 eV with a mass range from 41 to 450 m / z.
[035] Assim, a identificação dos compostos é realizada através da comparação dos padrões de fragmentação dos compostos da amostra com os presentes na base de dados Wiley (NIST versão 2.0, 2005) e na literatura, ou ainda, pela comparação do Índice de retenção, calculado com base nos tempos de retenção de hidrocarbonetos padrões (alcanos de C7-C40), com o índice de retenção presentes na literatura (ADAMS R. P. Identification of essential oil components by gas chromatography/mass spectroscopy. 4th ed. ed. Carol Stream: Allured Pub. Corp, 2007) .[035] Thus, the identification of compounds is carried out by comparing the fragmentation patterns of the sample compounds with those present in the Wiley database (NIST version 2.0, 2005) and in the literature, or even by comparing the Retention Index , calculated based on the standard hydrocarbon retention times (C7-C40 alkanes), with the retention index present in the literature (ADAMS RP Identification of essential oil components by gas chromatography / mass spectroscopy. 4th ed. ed. Carol Stream: Allured Pub. Corp, 2007).
[036] Por fim, na etapa f é realizada a quantificação relativa dos compostos presentes nas frações obtidas. Para tal, em um cromatógrafo gasoso com detecção por ionização de chama (FID), utiliza-se uma coluna com fase estacionária contendo 5% difenil e 95% dimetilpolisiloxano (por exemplo, OV-5ms, AT-5ms, BP-5ms, HP-5ms, HP-5ms UI ou outra similar) (30 m x 0,25 mm x 0,25 pm), em que como gás de arraste utiliza-se nitrogênio com fluxo 1 mL/min; volume de injeção de 1 pL; temperatura do injetor de 220 °C; e injeção realizada em modo splitless. Ainda, a temperatura do forno é programada para 50 °C por 2 minutos, uma rampa de 50[036] Finally, in step f, the relative quantification of the compounds present in the obtained fractions is performed. For this purpose, in a gas chromatograph with flame ionization detection (FID), a column with a stationary phase containing 5% diphenyl and 95% dimethylpolysiloxane (for example, OV-5ms, AT-5ms, BP-5ms, HP is used) -5ms, HP-5ms UI or similar) (30 mx 0.25 mm x 0.25 pm), in which carrier gas is used with nitrogen at a flow rate of 1 mL / min; injection volume of 1 pL; injector temperature of 220 ° C; and injection performed in splitless mode. In addition, the oven temperature is programmed to 50 ° C for 2 minutes, a ramp of 50
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12/24 °C a 310 °C aumentando 3 °C por minutos e espera a 310 °C por 20 minutos.12/24 ° C to 310 ° C increasing 3 ° C for minutes and waiting at 310 ° C for 20 minutes.
[037][037]
Assim, para o cálculo de porcentagem dos compostos presentes na amostra é utilizada a área total integrada do cromatograma, em que através do referido processo obtêm-se 91,47 % a 96,14 % de teor do composto trans-nerolidol, e 78,63 a 84,89 % de teor do composto espatulenol.Thus, to calculate the percentage of compounds present in the sample, the total integrated area of the chromatogram is used, in which through this process 91.47% to 96.14% of the content of the trans-nerolidol compound are obtained, and 78, 63 to 84.89% content of the spatulenol compound.
[038] Para avaliar o potencial do processo de purificação do óleo essencial de Baccharis dracunculifolia D.C. para obtenção de frações contendo trans-nerolidol e espatulenol da presente invenção, a seguir são apresentados uma exemplificação da invenção e os resultados dos testes realizados.[038] To evaluate the potential of the purification process of the essential oil of Baccharis dracunculifolia D.C. for obtaining fractions containing trans-nerolidol and spatulenol of the present invention, the following is an example of the invention and the results of the tests carried out.
Exemplos da invenção e testes realizados:Examples of the invention and tests carried out:
[039][039]
Vale ressaltar que, com o intuito de exemplificar a invenção, o presente processo foi realizado em cromatografia à média pressão. Todavia, é oportuno ressaltar que a presente invenção não é limitada pelos referidos exemplos, podendo ser facilmente adaptada à grande escala para reprodução industrial.It is worth mentioning that, in order to exemplify the invention, the present process was performed in medium pressure chromatography. However, it is worth noting that the present invention is not limited by the aforementioned examples, but can be easily adapted to the large scale for industrial reproduction.
[040][040]
A Figura 1 apresenta um fluxograma de uma exemplificação do processo de extração e fracionamento do óleo essencial de Baccharis dracunculifolia D.C. para obtenção de frações com alto teor de trans-nerolidol e espatulenol.Figure 1 presents a flowchart of an example of the extraction and fractionation process of the essential oil of Baccharis dracunculifolia D.C. to obtain fractions with a high content of trans-nerolidol and spatulenol.
[041][041]
Conforme mostrado na Figura 1, primeiramente, as folhas frescas (870 g) de Baccharis dracunculifolia D.C. foram hidrodestiladas por 4 horas obtendo um rendimento de 0,37 % de óleo essencial (3,2 g).As shown in Figure 1, first, the fresh leaves (870 g) of Baccharis dracunculifolia D.C. were hydrodistilled for 4 hours obtaining a yield of 0.37% of essential oil (3.2 g).
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13/24 [042] A identificação dos compostos presentes no óleo essencial foi realizada em um equipamento GC-MS ion trap (Varian Saturno 2100T), utilizando-se a coluna OV-5ms Bonded (30 m x 0,25 mm x 0,25 pm) (Ohio Valley Specialty) .13/24 [042] The identification of the compounds present in the essential oil was performed in a GC-MS ion trap equipment (Varian Saturno 2100T), using the OV-5ms Bonded column (30 mx 0.25 mm x 0.25 pm) (Ohio Valley Specialty).
[043] Foi utilizado o hélio como gás de arraste com um fluxo de 1 mL/minuto e o volume de injeção de 1 pL. A temperatura do injetor foi 220 °C e a injeção foi realizada em modo splitless. A temperatura do forno programada foi de 50 °C por 2 minutos, uma rampa de 50 °C a 310 °C aumentando 3 °C por minuto e espera a 310 °C por 20 minutos. O espectro de massas foi gerado em modo Scan a 70 eV com faixa de massas de 41 a 450 m/z.[043] Helium was used as carrier gas with a flow rate of 1 mL / minute and an injection volume of 1 pL. The injector temperature was 220 ° C and the injection was performed in splitless mode. The programmed oven temperature was 50 ° C for 2 minutes, a ramp from 50 ° C to 310 ° C increasing by 3 ° C per minute and waiting at 310 ° C for 20 minutes. The mass spectrum was generated in Scan mode at 70 eV with a mass range from 41 to 450 m / z.
[044] A identificação dos compostos foi realizada através da comparação dos padrões de fragmentação dos compostos da amostra com os presentes na base de dados Wiley (NIST versão 2.0, 2005) e na literatura, ou ainda, pela comparação do índice de retenção, calculado com base nos tempos de retenção de hidrocarbonetos padrões (alcanos de C7-C40), com o índice de retenção presentes na literatura (ADAMS R. P. Identification of essential oil components by gas chromatography/mass spectroscopy. 4th ed. ed. Carol Stream: Allured Pub. Corp, 2007) .[044] The identification of the compounds was performed by comparing the fragmentation patterns of the sample compounds with those present in the Wiley database (NIST version 2.0, 2005) and in the literature, or by comparing the retention index, calculated based on standard hydrocarbon retention times (C7-C40 alkanes), with the retention index present in the literature (ADAMS RP Identification of essential oil components by gas chromatography / mass spectroscopy. 4th ed. ed. Carol Stream: Allured Pub Corp, 2007).
[045] Assim, os constituintes majoritários encontrados no óleo essencial de alecrim do campo foram o monoterpeno limoneno (11,49 %), e dois sesquiterpenos, o trans-nerolidol (36,64 %) e o espatulenol (10,28 %) (Tabela 1).[045] Thus, the major constituents found in the essential oil of rosemary from the field were monoterpene limonene (11.49%), and two sesquiterpenes, trans-nerolidol (36.64%) and spatulenol (10.28%) (Table 1).
Tabela 1 - Composição química do óleo essencial de Baccharis dracunculifolia D.C.Table 1 - Chemical composition of the essential oil of Baccharis dracunculifolia D.C.
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tR(min)= tempo de retenção dos compostos em minutos; AI cal= índice aritmético calculado com base nos tempos de retenção de padrões de alcanos (C7-40); AI Lit= índice aritmético obtido da literatura (R.P. Adams: Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry.” Allured publishing, Carol Stream , 2007); % Relativa= porcentagem de área total integrada do cromatograma; MM= massa molecular de compostos não identificados; tr= compostos com % relativa <0,1.tR (min) = compound retention time in minutes; AI cal = arithmetic index calculated based on the retention times of alkane patterns (C7-40); AI Lit = arithmetic index obtained from the literature (RP Adams: Identification of Essential Oil Components by Gas Chromatography / Mass Spectrometry. ”Allured publishing, Carol Stream, 2007); Relative% = percentage of total integrated area of the chromatogram; MM = molecular mass of unidentified compounds; tr = compounds with relative% <0.1.
[046] Nos cromatogramas obtidos no GC-FID e no GCMS (Figura 2) estão numerados os compostos identificados no óleo essencial de Baccharis dracunculifolia D.C.[046] In the chromatograms obtained in GC-FID and GCMS (Figure 2) the compounds identified in the essential oil of Baccharis dracunculifolia D.C. are numbered
[047] Posteriormente, o óleo essencial (1,8 g) de Baccharis dracunculifolia D.C. foi fracionado por cromatografia Flash utilizando o equipamento Biotage IsoleraTM com um cartucho de silica de 100g (SNAP KP-SIL). Foram utilizados os solventes n-hexano (A) e acetato de etila (B) como fase móvel e um fluxo de 50 mL/minuto. O gradiente utilizado para a separação foi: 100 % de A (de 0 a 3 minutos) , 0 a 10 % de B (3 a 12 minutos), 10 a 20 % de B (12 a 18 minutos) e 20 a 35 % de B (18 a 21 minutos). As frações foram coletadas, em volumes máximos de 20 mL, conforme a detecção de substâncias nos comprimentos de onda 254 nm e 280 nm, obtendo-se um total de 140 frações (Figura 3).[047] Subsequently, the essential oil (1.8 g) of Baccharis dracunculifolia DC was fractionated by Flash chromatography using the Biotage Isolera TM equipment with a 100g silica cartridge (SNAP KP-SIL). The solvents n-hexane (A) and ethyl acetate (B) were used as the mobile phase and a flow rate of 50 mL / minute. The gradient used for the separation was: 100% A (0 to 3 minutes), 0 to 10% B (3 to 12 minutes), 10 to 20% B (12 to 18 minutes) and 20 to 35% from B (18 to 21 minutes). The fractions were collected, in maximum volumes of 20 mL, according to the detection of substances in the wavelengths 254 nm and 280 nm, obtaining a total of 140 fractions (Figure 3).
[048] A identificação dos compostos presentes nas frações ativas foi realizada conforme a identificação dos componentes do óleo essencial detalhada anteriormente.[048] The identification of the compounds present in the active fractions was carried out according to the identification of the essential oil components detailed previously.
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16/24 [049] A quantificação relativa dos compostos presentes nas amostras foi realizada em um equipamento GC Shlmadzu 2010 com injetor AOC 5000 e detector de ionização de chama (FID), utilizando a mesma coluna utilizada para as análises no espectro de massas GC-MS. O gás de arraste utilizado foi nitrogênio com fluxo 1 mL/min e o volume de injeção foi de 1 pL. A temperatura programada do forno e a temperatura do injetor foram as mesmas utilizadas nas análises do GC-MS. A injeção foi feita em modo splltless. Para o cálculo de porcentagem dos compostos presente na amostra foi utilizada a área total integrada do cromatograma.16/24 [049] The relative quantification of the compounds present in the samples was performed in a GC Shlmadzu 2010 equipment with AOC 5000 injector and flame ionization detector (FID), using the same column used for the analyzes in the GC-mass spectrum MS. The carrier gas used was nitrogen with flow 1 mL / min and the injection volume was 1 pL. The programmed oven temperature and the injector temperature were the same used in the GC-MS analyzes. The injection was done in splltless mode. To calculate the percentage of compounds present in the sample, the total integrated area of the chromatogram was used.
[050] Entre o total de frações obtidas, as frações 89 - 92 foram as que apresentaram maior teor (de 91,47 % a 96,14 %) do composto de interesse, trans-nerolidol, totalizando 395,8 mg e recuperação de 22% do total de amostra. Esse resultado de recuperação é considerado alto uma vez que a porcentagem de trans-nerolidol presente na amostra de óleo essencial é de 36,64 % (Tabela 1). A fração 92, que apresentou o composto trans-nerolidol com teor de 91,47 % (GC-FID) foi utilizada no preparo das emulsões para a realização dos testes de atividade antibacteriana. Os constituintes identificados nesta fração estão descritos na Tabela 2 e o espectro de massas (GC-MS) obtido do composto trans-nerolidol está apresentado na Figura 4.[050] Among the total fractions obtained, fractions 89 - 92 were those with the highest content (from 91.47% to 96.14%) of the compound of interest, trans-nerolidol, totaling 395.8 mg and recovery of 22% of the total sample. This recovery result is considered high since the percentage of trans-nerolidol present in the essential oil sample is 36.64% (Table 1). The fraction 92, which presented the trans-nerolidol compound with a content of 91.47% (GC-FID), was used in the preparation of the emulsions to perform the antibacterial activity tests. The constituents identified in this fraction are described in Table 2 and the mass spectrum (GC-MS) obtained from the trans-nerolidol compound is shown in Figure 4.
Tabela 2 - Composição química da Fração 92 (OE deTable 2 - Chemical composition of Fraction 92 (OE of
Baccharis dracuncullfolla D.C)Baccharis dracuncullfolla D.C)
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tR(min)= tempo de retenção dos compostos em minutos; AI cal= índice aritmético calculado com base nos tempos de retenção de padrões de alcanos (C7-40); AI Lit= índice aritmético obtido da literatura (R.P. Adams: Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry.” Allured publishing, Carol Stream , 2007); % Relativa= porcentagem de área total integrada do cromatograma.tR (min) = compound retention time in minutes; AI cal = arithmetic index calculated based on the retention times of alkane patterns (C7-40); AI Lit = arithmetic index obtained from the literature (RP Adams: Identification of Essential Oil Components by Gas Chromatography / Mass Spectrometry. ”Allured publishing, Carol Stream, 2007); Relative% = percentage of total integrated area of the chromatogram.
[051] A combinação das frações 99 - 102 (total de 139,4 mg) (Figura 3), que apresentaram o composto espatulenol em maiores proporções, foi re-fracionada no Biotage IsoleraTM com um cartucho de sílica de 50g (SNAP KP-SIL) . Foram utilizados os solventes ciclohexano (A) e diclorometano (B) como fase móvel e um fluxo de 50 mL/minuto. O gradiente utilizado para a separação foi: 60 % de B (de 0 a 4 minutos) , 60 a 100 % de B (4 a 18 minutos), 100 % de B (18 a 29 minutos). As frações foram coletadas, em volumes máximos de 15 mL, conforme a detecção de substâncias nos comprimentos de onda 210 nm e 254 nm, obtendo as frações R51 a R54, com maior teor de espatulenol (de 78,63 a 84,89 %), totalizando, 48,5 mg e recuperação de 2,7% da amostra inicial de óleo essencial (1,8 g). A fração R51 que apresentou o composto espatulenol com um teor de 78,63% (GC-FID) foi utilizada para o preparo de emulsões para avaliar a atividade antibacteriana. A composição química desta fração está descrita na Tabela 3 e o espectro de massas (GC-MS) obtido do composto espatulenol está disposto na Figura 5.[051] The combination of fractions 99 - 102 (total of 139.4 mg) (Figure 3), which presented the spatulenol compound in greater proportions, was re-fractionated in Biotage Isolera TM with a 50g silica cartridge (SNAP KP -SIL). The solvents cyclohexane (A) and dichloromethane (B) were used as a mobile phase and a flow rate of 50 mL / minute. The gradient used for the separation was: 60% B (0 to 4 minutes), 60 to 100% B (4 to 18 minutes), 100% B (18 to 29 minutes). The fractions were collected, in maximum volumes of 15 mL, according to the detection of substances at wavelengths 210 nm and 254 nm, obtaining fractions R51 to R54, with a higher content of spatulenol (from 78.63 to 84.89%) , totaling 48.5 mg and recovery of 2.7% of the initial essential oil sample (1.8 g). The fraction R51 that presented the compound spatulenol with a content of 78.63% (GC-FID) was used for the preparation of emulsions to evaluate the antibacterial activity. The chemical composition of this fraction is described in Table 3 and the mass spectrum (GC-MS) obtained from the spatulenol compound is shown in Figure 5.
Tabela 3 - Composição química da Fração R51 (OE deTable 3 - Chemical composition of Fraction R51 (OE of
Baccharis dracunculifolia D.C)Baccharis dracunculifolia D.C)
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tR(min)= tempo de retenção dos compostos em minutos; AI cal= índice aritmético calculado com base nos tempos de retenção de padrões de alcanos (C7-40); AI Lit= índice aritmético obtido da literatura (R.P. Adams: Identification of Essential Oil Components by Gas Chromatography/ Mass Spectrometry.” Allured publishing, Carol Stream , 2007); MM= massa molecular de compostos não identificados; % Relativa= porcentagem de área total integrada do cromatograma.tR (min) = compound retention time in minutes; AI cal = arithmetic index calculated based on the retention times of alkane patterns (C7-40); AI Lit = arithmetic index obtained from the literature (RP Adams: Identification of Essential Oil Components by Gas Chromatography / Mass Spectrometry. ”Allured publishing, Carol Stream, 2007); MM = molecular mass of unidentified compounds; Relative% = percentage of total integrated area of the chromatogram.
- Testes de atividade antibacteriana:- Antibacterial activity tests:
[052] O método de microdiluição foi utilizado para determinar a concentração inibitória mínima (CMI) e a concentração bactericida mínima (CBM) do OE de Baccharis dracunculifolia D.C. (Asteraceae) e de duas frações deste óleo, contendo principalmente trans-nerolidol ou espatulenol, contra Staphylococcus aureus e Streptococcus agalactiae.[052] The microdilution method was used to determine the minimum inhibitory concentration (CMI) and minimum bactericidal concentration (CBM) of the OE of Baccharis dracunculifolia DC (Asteraceae) and two fractions of this oil, containing mainly trans-nerolidol or espatulenol, against Staphylococcus aureus and Streptococcus agalactiae.
[053] O teste foi realizado utilizando Staphylococcus aureus e Streptococcus agalactiae isolados de mastite bovina, bem como, cepas S. aureus resistente à vancomicina (VRSA) Mu50, S. aureus resistente à meticilina (MRSA) ATCC43300, S. aureus sensível a meticilina (MSSA) ATCC29213 e S. agalactiae ATCC13813.[053] The test was performed using Staphylococcus aureus and Streptococcus agalactiae isolated from bovine mastitis, as well as vancomycin-resistant S. aureus (VRSA) strains Mu50, methicillin-resistant S. aureus (MRSA) ATCC43300, methicillin-sensitive S. aureus (MSSA) ATCC29213 and S. agalactiae ATCC13813.
[054] As amostras foram previamente pesadas e uma solução de polissorbato 80 0,03% (p/v) foi adicionada, obtendo as concentrações desejadas. A solução de polissorbato 80 0,03% (p/v) foi preparada com o meio de cultivo utilizado nos testes de atividade antibacteriana e esterilizada por filtração com membrana RC 0,22 pm. Para formar a emulsão, as amostras foram homogeneizadas por 1 minuto em vórtex, sonicadas em banho de ultrassom por 10 minutos e homogeneizadas novamente em vórtex por 1 minuto. O caldo Mueller-Hinton (MH) foi o meio de cultivo utilizado[054] The samples were previously weighed and a solution of polysorbate 80 0.03% (w / v) was added, obtaining the desired concentrations. The 0.03% (w / v) polysorbate 80 solution was prepared with the culture medium used in the antibacterial activity tests and sterilized by filtration with a 0.22 pm RC membrane. To form the emulsion, the samples were homogenized for 1 minute in a vortex, sonicated in an ultrasound bath for 10 minutes and homogenized again in a vortex for 1 minute. The Mueller-Hinton broth (MH) was the culture medium used
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19/24 para o preparo da solução de polissorbato 80 0,03% (p/v) utilizada nos testes com S. aureus, e para os testes com S. agalactiae utilizou-se o caldo Brain Heart Infusion (BHI).19/24 for the preparation of the polysorbate 80 0.03% (w / v) solution used in the tests with S. aureus, and for the tests with S. agalactiae, the Brain Heart Infusion (BHI) broth was used.
[055] A solução de polissorbato 80 0,03% (p/v) (50 pL) foi adicionada em cada poço da microplaca de 96 poços. Um volume de 100 pL da emulsão previamente preparada foi adicionado ao primeiro poço e em seguida foi realizada a diluição seriada de cada amostra.[055] The 0.03% (w / v) polysorbate 80 solution (50 pL) was added to each well of the 96-well microplate. A volume of 100 pL of the previously prepared emulsion was added to the first well and then serial dilution of each sample was performed.
[056] A concentração das suspensões bacterianas com 24 horas de crescimento a 35 °C (± 2 °C) foi ajustada para aproximadamente 1 x 108 unidades formadoras de colônias por mililitro (UFC/mL), (correspondente à 0,5 na escala de McFarland). Em seguida, essas suspensões foram diluídas, obtendo uma concentração final de 105 UFC/mL para S. aureus e 106 UFC/mL para S. agalactiae, e adicionadas (50 pL por poço) à microplaca. A microplaca foi incubada em estufa por 24 horas a 35 °C (± 2 °C).[056] The concentration of bacterial suspensions with 24 hours of growth at 35 ° C (± 2 ° C) was adjusted to approximately 1 x 10 8 colony-forming units per milliliter (CFU / mL), (corresponding to 0.5 in McFarland scale). Then, these suspensions were diluted, obtaining a final concentration of 10 5 CFU / mL for S. aureus and 10 6 CFU / mL for S. agalactiae, and added (50 pL per well) to the microplate. The microplate was incubated in an oven for 24 hours at 35 ° C (± 2 ° C).
[057] As concentrações finais das emulsões avaliadas no teste de atividade antibacteriana foram: a) OE de Baccharis dracunculifolia D.C., de 0,093 a 0,75 mg/mL (para S. aureus) e de 0,046 a 0,375 mg/mL (para S. agalactiae) ; b) fração 92 contendo trans-nerolidol, de 0,046 a 0,375 mg/mL (para S. aureus) e de 0,023 a 0,187 mg/mL (para S. agalactiae); b) fração R51 contendo espatulenol, de 0,046 a 0,375 mg/mL (para S. aureus) e de 0,023 a 0,187 mg/mL (para S. agalactiae). A concentração final de polissorbato 80 no poço foi de 0,015% (p/v). Os testes foram realizados em triplicata.[057] The final concentrations of the emulsions assessed in the antibacterial activity test were: a) Baccharis dracunculifolia DC E, 0.093 to 0.75 mg / mL (for S. aureus) and 0.046 to 0.375 mg / mL (for S agalactiae); b) fraction 92 containing trans-nerolidol, from 0.046 to 0.375 mg / ml (for S. aureus) and from 0.023 to 0.187 mg / ml (for S. agalactiae); b) fraction R51 containing spatulenol, from 0.046 to 0.375 mg / mL (for S. aureus) and from 0.023 to 0.187 mg / mL (for S. agalactiae). The final concentration of polysorbate 80 in the well was 0.015% (w / v). The tests were performed in triplicate.
[058] Após o período de incubação, uma alíquota (5 pL) foi retirada de cada poço e inoculada em placas de ágar[058] After the incubation period, an aliquot (5 pL) was removed from each well and inoculated on agar plates
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BHI para determinar a CBM. A concentração inibitória mínima foi determinada após 3 horas da adição de 50 pL da solução aquosa de 3 mg/mL de TTC (cloreto de 2,3,5trifeniltetrazólio) utilizado no teste com S. aureus, e da solução aquosa de 1,5 mg/mL de MTT (Brometo de 3-(4,5dimetil-2-tiazolil)-2,5-difenil-2H-tetrazólio) para o teste com S. agalactiae. A CIM foi definida como a menor concentração em que as amostras inibiram visualmente o crescimento bacteriano.BHI to determine CBM. The minimum inhibitory concentration was determined 3 hours after the addition of 50 pL of the 3 mg / mL aqueous solution of TTC (2,3,5 triphenyltetrazolium chloride) used in the S. aureus test, and the 1.5 mg aqueous solution / mL MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) for the test with S. agalactiae. MIC was defined as the lowest concentration at which the samples visually inhibited bacterial growth.
[059] Uma solução polissorbato 80 0,015% (p/v) foi utilizada como controle negativo. A solução aquosa de ampicilina (Sigma-Aldrich, ref. A6140) também preparada com o meio de cultivo foi utilizada como controle positivo nas concentrações, de 2 a 16 pg/mL (para S. aureus), e de 0,125 a 1 pg/mL (para S. agalactiae).[059] A 0.015% polysorbate 80 solution (w / v) was used as a negative control. The aqueous solution of ampicillin (Sigma-Aldrich, ref. A6140) also prepared with the culture medium was used as a positive control in the concentrations, from 2 to 16 pg / mL (for S. aureus), and from 0.125 to 1 pg / mL (for S. agalactiae).
[060] Na Tabela 4 estão apresentados os resultados de CIM e CBM das emulsões preparadas com o OE B. dracunculifolia D.C. e das frações contendo trans-nerolidol e espatulenol, contra Staphylococcus aureus e Streptococcus agalactiae. A emulsão preparada com o OE do B. dracunculifolia D.C. apresentou atividade antibacteriana contra Staphylococcus aureus e Streptococcus agalactiae associados à mastite bovina com MIC de ^0,093 a 0,75 mg/mL. As emulsões contendo a fração 92 (trans-nerolidol) e a fração R51 (espatulenol) apresentaram MIC de 0,093 a 0,375 mg/mL (Tabela 4). Observou-se a inibição do crescimento bacteriano de patógenos isolados de mastite bovina, resistentes a antimicrobianos utilizados no tratamento desta doença, e das cepas S. aureus ATCC 43300 (MRSA) e Mu 50 (VRSA), que apresentam resistência a mais de uma classe de[060] Table 4 shows the results of MIC and CBM of emulsions prepared with OE B. dracunculifolia D.C. and fractions containing trans-nerolidol and spatulenol, against Staphylococcus aureus and Streptococcus agalactiae. The emulsion prepared with the OE of B. dracunculifolia D.C. showed antibacterial activity against Staphylococcus aureus and Streptococcus agalactiae associated with bovine mastitis with MIC of ^ 0.093 to 0.75 mg / mL. Emulsions containing fraction 92 (trans-nerolidol) and fraction R51 (spatulenol) showed MICs from 0.093 to 0.375 mg / mL (Table 4). Bacterial growth inhibition was observed in isolated pathogens isolated from bovine mastitis, resistant to antimicrobials used in the treatment of this disease, and the S. aureus ATCC 43300 (MRSA) and Mu 50 (VRSA) strains, which are resistant to more than one class in
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21/24 antimicrobianos.21/24 antimicrobials.
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Tabela 4 - Concentração Mínima Inibitória (MIC) e Concentração Bactericida Mínima (MBC) das emulsões com OE do B. dracunculifolia D.C., fração 92 (trans-nerolidol) e fração R51 (espatulenol), contra bactérias patogênicas.Table 4 - Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of emulsions with B. dracunculifolia D.C. OE, fraction 92 (trans-nerolidol) and fraction R51 (spatulenol), against pathogenic bacteria.
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a não apresentou resistência aos antimicrobianos avaliados; b Avaliado com o método de difusão de disco (CLSI, 2016); AMP = Ampicilina 10 pg; CFL = Cefalotina 30 pg; CIP = Ciprofloxacina 5 pg; GEN = Gentamicina 10 pg; PEN = Penicilina 10 UI; OXA= Oxacilina (avaliado com Cefoxitina 30 pg); VAN = Vancomicina 30 pg a did not show resistance to the evaluated antimicrobials; b Evaluated using the disk diffusion method (CLSI, 2016); AMP = Ampicillin 10 pg; CFL = cephalothin 30 pg; CIP = Ciprofloxacin 5 pg; GEN = Gentamicin 10 pg; PEN = Penicillin 10 IU; OXA = Oxacillin (assessed with Cefoxitin 30 pg); VAN = Vancomycin 30 pg
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24/24 [061] Portanto, o processo de purificação do óleo essencial da planta B. dracunculifolia D.C. aqui descrito, é uma alternativa rápida e de baixo custo para obtenção de frações com alto teor de trans-nerolidol (de 91,47% a 96,14%) e espatulenol (de 78,63 a 84,89%).24/24 [061] Therefore, the purification process of the essential oil of the B. dracunculifolia DC plant described here, is a fast and low cost alternative for obtaining fractions with a high content of trans-nerolidol (from 91.47% to 96.14%) and spatulenol (from 78.63 to 84.89%).
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