Extração de bioativos derivados de Cannabis via fluidos pressurizados

Abstract

As plantas do genero Cannabis possuem substancias especificas chamadas canabinoides. O canabidiol (CBD) e o ƒ'9-tetrahidrocanabinol (ƒ'9-THC) sao canabinoides que possuem importantes aplicabilidades medicinais. Altos teores de canabinoides sao encontrados na resina de flores de plantas femeas nao polinizadas. O CBD e o ƒ'9-THC estao em baixas concentracoes em flores recem-colhidas, mas sao potencializados com o aquecimento de uma amostra. O canhamo e uma variedade de Cannabis com baixos teores de canabinoides e suas sementes sao ricas em acidos graxos poli-insaturados. A obtencao destes principios ativos com alta pureza e livre de solventes sao amplamente requisitadas. Este trabalho tem como objetivo aplicar a tecnologia de fluidos pressurizados analisando a variacao de parametros para otimizar a obtencao de extratos com propriedades bioativas a partir de flores e sementes de Cannabis. Foram analisadas tres variedades de flores em parceria com a Universidade da Republica do Uruguai, utilizando um metodo cromatografico certificado. Foi avaliado o comportamento termico das amostras de flores a 90, 100 e 140 oC, a umidade, bem como a influencia do tamanho de particulas por microscopia eletronica de varredura (MEV). As extracoes de flores foram feitas com dioxido de carbono supercritico (CO2-SC) sendo comparadas as metodologias de pre-aquecimento das amostras, de uso de co-solvente e de extracao fracionada. Avaliou-se a atividade anti-tumoral dos extratos por um metodo quantitativo colorimetrico chamado MTT. Devido ao grande interesse medicinal no CBD foi analisado o equilibrio solido-fluido deste com o CO2-SC a fim de determinar condicoes favoraveis de manipulacao. Foi utilizado o metodo sintetico visual na faixa de temperatura e fracao molar de 45 a 65 oC e 2 a 3 (10-4), respectivamente. Paralelamente foi analisada a composicao centesimal de sementes descascadas de canhamo, bem como extracoes com CO2-SC e propano pressurizado, sendo ainda avaliada nos extratos a composicao de acidos graxos, tocoferois totais e ƒÒ-caroteno, bem como os indices de acidez e umidade. Os extratos de flores obtidos com co-solvente apresentaram maiores rendimentos, porem menores concentracoes de canabinoides. Por outro lado nos extratos com pre-aquecimento foi possivel obter elevadas e distintas concentracoes de CBD e ƒ'9-THC. A extracao fracionada para flores tambem se apresentou eficiente. Os melhores resultados de atividade anti-tumoral foram relacionados ao cancer de utero. As analises de equilibrio de fase apresentaram baixos desvios e bons ajustes. O propano pressurizado se demostrou uma alternativa interessante para a obtencao de oleo das sementes frente ao CO2-SC, visto que foi necessaria uma menor quantidade de solvente, obteve-se maiores teores de tocoferois totais e ƒÒ-caroteno, menores indices de umidade e acidez, bem como os mesmos elevados teores de acidos graxos poli-insaturados.

Plants of the genus Cannabis have specific substances called cannabinoids. Cannabidiol (CBD) and ƒ'9-tetrahydrocannabinol (ƒ'9-THC) are cannabinoids that have important medicinal applications. High levels of cannabinoids are found in the flower resin of non-pollinated female plants. CBD and ƒ'9-THC are at low concentrations in freshly harvested flowers, but are potentiated by heating a sample. Hemp is a variety of Cannabis with low levels of cannabinoids and their seeds are rich in polyunsaturated fatty acids. Obtaining these active substances with high purity and solvent-free are widely required. This work aims to apply pressurized fluid technology analyzing the variation of parameters to optimize the obtaining of extracts with bioactive properties from Cannabis flowers and seeds. Three varieties of flowers were analyzed in partnership with the University of the Republic of Uruguay using a certified chromatographic method. The thermal behavior of flower samples at 90, 100 and 140 oC, moisture, as well as the influence of particle size by scanning electron microscopy (SEM) was evaluated. The flower extractions were made with supercritical carbon dioxide (SC-CO2), comparing the sample pre-heating, co-solvent and fractionation extraction methodologies. The antitumor activity of the extracts was evaluated by a quantitative colorimetric method called MTT. Due to the great medicinal interest in the CBD the solid-fluid equilibria of this with the SC-CO2 was analyzed in order to determine favorable conditions of manipulation. The visual synthetic method was used in the temperature and molar fraction range from 45 to 65 oC and 2 a 3 (10-4), respectively. In parallel, the centesimal composition of hulled hemp seeds was analyzed, as well as SC-CO2 and pressurized propane extractions. Compositions of fatty acids, total tocopherols and ƒÒ-carotene were evaluated in the extracts, as well as acidity and moisture indexes. The extracts of flowers obtained with co-solvent showed higher yields, but lower concentrations of cannabinoids. However in extracts with preheating it was possible to obtain high and different concentrations of CBD and ƒ'9-THC. The fractionated extraction for flowers was also efficient. The best results of anti-tumor activity were related to cervical cancer. The phase equilibrium analyzes showed low deviations and good adjustments. The pressurized propane showed an interesting alternative to obtain oil of the seeds compared to SC-CO2, since a smaller amount of solvent was required, higher contents of total tocopherols and ƒÒ-carotene, lower moisture and acidity indexes, as well as the same high levels of polyunsaturated fatty acids.