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A espécie Coriandrum sativum L., vulgarmente conhecida como coentro, é frequentemente usada na alimentação, mas também em aplicações medicinais, na cosmética e perfumaria. É uma fonte de polifenóis e outros fitoquímicos, relacionados com a sua elevada actividade antioxidante e com a sua utilização no combate a indigestões, reumatismo e na prevenção dos danos provocados pela peroxidação lipídica. A cultura de células vegetais é um meio para estudar e produzir alguns compostos activos nomeadamente, voláteis, polifenóis e outros metoblitos secundários. No presente trabalho, foram germinadas sementes de coentro em condições de cultura in vitro, após excisão dos meristemas das plântulas e inoculação em meio modificado MS contendo IBA e BAP. Após seis meses em cultura, diferenciaram-se duas linhas pela sua pigmentação: a linha A, apresentando uma elevada coloração púrpura e a linha B, com uma coloração verde. A taxa de multiplicação dos meristemas foi de 50% para ambas as linhas após 3 semanas, apesar do padrão de crescimento não ser o mesmo. Estudou-se a taxa relativa de crescimento relacionando massa fresca e seca das duas linhas. A linha A revelou um maior crescimento apesar da relação massa fresca/massa seca ter sido maior na linha B, o que pode ter sido devido a uma maior concentração de água na planta. No final da 4ª semana do ciclo de micropropagação, a linha A ainda mostrava crescimento activo, em oposição à linha B, que apresentava um declínio do crescimento no final da 3ª semana.
Foram estudadas amostras obtidas in vivo (partes aéreas e sementes) e as amostras obtidas in vitro (linhas A e B). Caracterizaram-se os voláteis (maioritariamente compostos terpénicos) presentes nas amostras após isolamento por hidrodestilação e análise por GC e GC-MS. O linalol foi o volátil predominante nas sementes (82%), seguido de γ-terpineno (4%), cânfora (3%) e geraniol (3%). O linalol estava também presente nas fracções voláteis das amostras obtidas in vitro, linhas A e B, e também nas partes aéreas obtidas in vivo, sempre em pequenas quantidades relativas (0,1%, 0,1% e 0,3%, respectivamente). O dodecanal (17%), o dodecanol (17%), o n-tetradecanol (15%) e o decanal (10%) foram os voláteis maioritários nas partes aéreas obtidas in vivo. O β-felandreno (37% em A, 45% em B), o terpinoleno (9% em ambos), o β-sesquifelandreno (4% em A, 6% em B) e o α-felandreno (2% em A, 3% em B) foram os voláteis maioritários identificados nas amostras obtidas in vitro, linhas A e B. Apesar da coloração púrpura observada nas plantas da linha A, o seu perfil volátil foi quantitativa e qualitativamente muito semelhante ao da linha B. A composição de voláteis nas amostras obtidas in vitro foi qualitativamente semelhante à das sementes e muito diferente das amostras obtidas in vivo.
Quantificaram-se também os compostos antioxidantes lipofílicos (tocoferóis, carotenóides e clorofilas) e hidrofílicos (açúcares, ácido ascórbico, fenóis, flavonóis e antocianinas) presentes nas amostras. Além disso, as suas propriedades antioxidantes foram avaliadas atráves da actividade captadora de radicais livres, poder redutor e inibição da peroxidação lipídica. As partes aéreas obtidas in vivo mostraram maior actividade antioxidante principalmente devido aos seus níveis mais elevados de compostos hidrofílicos. Pelo contrário, as amostras obtidas in vitro, em especial a linha A, apresentaram maior concentração em compostos lipofílicos mas um perfil distinto quando comparado com as partes aéreas obtidas in vivo. As linhas A e B revelaram ausência de β-caroteno, β- e δ-tocoferóis, um decréscimo em α-tocoferol e um aumento em ү-tocoferol e clorofilas, comparativamente às amostras obtidas in vivo.
Foi ainda realizada uma análise detalhada dos compostos fenólicos individuais presentes nas amostras obtidas in vivo e in vitro. As partes aéreas obtidas in vivo, mostraram conter derivados de quercetina como flavonóides maioritários, em especial 3-O-rutinósido de quercetina (3296 mg/kg dw). As sementes revelaram apenas a presença de ácidos fenólicos e derivados, com o hexósido de cafeoil N-triptofano como maioritário (45,33 mg/kg dw). As amostras obtidas in vitro demonstraram também uma grande variedade de polifenóis, sendo a apigenina C-glucosilada o composto maioritário (2983 mg/kg dw). As antocianinas foram encontradas, exclusivamente, n linha A o que está certamente relacionado com a sua pigmentação púrpura; a peonidin-3-O-feruloilglucósido-5-O-glucósido foi a antocianina maioritária (1,70 μg/kg dw). Recorrendo a uma análise molecular e, utilizando as partes aéreas do indivíduo in vivo como controlo externo, foi possível distinguir os indivíduos crescidos in vitro, linhas A e B, com base em diferenças fenotípicas. O cluster ISSR não mostrou correlação entre as amostras obtidas in vivo e in vitro (índice de similaridade inferior a 0,30). Os indivíduos obtidos in vitro revelaram um maior índice de similaridade (0,70), o que indica que as variações genéticas são baixas e não podem explicar as diferenças fenotípicas encontradas entre os dois clones. A cultura in vitro pode ser útil para explorar as potencialidades das plantas para aplicações industriais, controlando as condições ambientais para produzir maior quantidade de alguns produtos bioactivos. Pode ser também utilizada para explorar novas potencialidades industriais, farmacêuticas e medicinais, nomeadamente a produção de metabolitos secundários tais como voláteis e compostos fenólicos.
Coriandrum sativum L. (known as coriander) is commonly used for medicinal purposes, food applications, cosmetics and perfumes. It is a source of a variety of polyphenols and other phytochemicals, related to its high antioxidant activity and to its use for indigestion, rheumatism, and prevention of lipid peroxidation damage. Plant cell cultures are a mean to study or to produce some active metabolites such as volatiles, polyphenols and other secondary metabolites. In the present work, seeds of coriander were germinated in vitro and shoots were excised from the seedlings and inoculated in a modified MS medium containing IBA and BAP. After six months in culture, two stable clones were differentiated by their pigmentation: clone A, presenting a high purple coloration and clone B, with the normal green coloration. The rate of shoots multiplication was 50% for both clones after 3 weeks, although the growth pattern was not the same. The relative growth rate was studied relating fresh and dry weight of the two clones. Clone A plants revealed a higher growth although the relation fresh weight/dry weight was higher in clone B, which can be due to a higher water concentration on the plant. At the end of the 4th week of the microprogation cycle, clone A still showed an active growth, in opposition to clone B that declined in growth at the end of the 3th week. The studied samples were in vivo aerial parts and seeds, and in vitro clones A e B. The volatiles (mainly terpenic compounds) were characterized after isolation by hydrodistillation and analysis by GC and GC-MS. The seeds volatiles were dominated by linalool (82%), with γ-terpinene (4%), camphor (3%) and geraniol (3%) as other compounds. Linalool was present in the volatile fractions of clones A and B in vitro samples and of in vivo aerial parts, always in small relative amounts (0.1%, 0.1% and 0.3%, respectively). Dodecanal (17%), dodecanol (17%), n-tetradecanol (15%) and decanal (10%) were the dominant volatiles in the in vivo vegetative parts. β-Phellandrene (37% in A, 45% in B), terpinolene (9% in both), β-sesquiphellandrene (4% in A, 6% in B) and α-phellandrene (2% in A, 3% in B) were the major identified volatiles in clones A and B in vitro samples. Despite the purple coloration in clone A plants, the volatile profile was quantitative and qualitatively very similar to clone B. The volatiles composition of in vitro grown plants was qualitatively similar to the seeds volatiles and very different from the in vivo aerial parts volatiles. Lipophilic (tocopherols, carotenoids and chlorophylls) and hydrophilic (sugars, ascorbic acid, phenolics, flavonols and anthocyanins) compounds were quantified. Furthermore, the antioxidant properties were evaluated by free radical scavenging activity, reducing power and lipid peroxidation inhibition. In vivo aerial parts showed the highest antioxidant activity mainly due to its highest levels of hydrophilic compounds. Otherwise, in vitro samples, mainly clone A, gave the highest concentration in lipophilic compounds but a different profile when compared to the in vivo vegetative parts. Clones A and B revealed a lack of β -carotene, β- and δ-tocopherols, a decrease in α -tocopherol, and an increase in ү -tocopherol, and an increase in y-tocopherol and chlorophylls in comparison to the in vivo sample. A detailed analysis of individual phenolic compounds present in in vivo and in vitro grown samples, was also performed. In vivo aerial parts showed quercetin derivatives as the main flavonoids, and quercetin-3-O-rutinoside was the main polyphenol found in this part of coriander (3296 mg/kg dw). The seeds revealed only phenolic acids and derivatives, being caffeoyl N-tryptophan hexoside the most abundant phenolic derivative (45.33 mg/kg dw). In vitro samples also gave a high diversity of polyphenols, being C-glycosylated apigenin the main compound (2983 mg/kg dw). Anthocyanins were only found in clone A, which was certainly related to its purple pigmentation, and peonidin-3-O-feruloylglucoside-5-O-glucoside was the major anthocyanin found (1.70 μg/kg dw). With molecular analysis we could distinguish in vitro grown individuals of clone A and clone B based on phenotypic differences, using in vivo aerial parts individuals as an out group control. The ISSR cluster showed no correlation between in vivo and in vitro grown samples (similarity index below 0.30). In vitro individuals revealed a high similarity index (0.70), meaning that the genetic variation is low and could not explain the phenotypic differences found between the two clones. In vitro culture might be useful to explore the plants potentialities for industrial applications, controlling environmental conditions to produce higher amounts of some bioactive products. It can also be used to explore new industrial, pharmaceutical and medicinal potentialities, such as the production of secondary metabolites like volatiles and phenolic compounds.
Coriandrum sativum L. (known as coriander) is commonly used for medicinal purposes, food applications, cosmetics and perfumes. It is a source of a variety of polyphenols and other phytochemicals, related to its high antioxidant activity and to its use for indigestion, rheumatism, and prevention of lipid peroxidation damage. Plant cell cultures are a mean to study or to produce some active metabolites such as volatiles, polyphenols and other secondary metabolites. In the present work, seeds of coriander were germinated in vitro and shoots were excised from the seedlings and inoculated in a modified MS medium containing IBA and BAP. After six months in culture, two stable clones were differentiated by their pigmentation: clone A, presenting a high purple coloration and clone B, with the normal green coloration. The rate of shoots multiplication was 50% for both clones after 3 weeks, although the growth pattern was not the same. The relative growth rate was studied relating fresh and dry weight of the two clones. Clone A plants revealed a higher growth although the relation fresh weight/dry weight was higher in clone B, which can be due to a higher water concentration on the plant. At the end of the 4th week of the microprogation cycle, clone A still showed an active growth, in opposition to clone B that declined in growth at the end of the 3th week. The studied samples were in vivo aerial parts and seeds, and in vitro clones A e B. The volatiles (mainly terpenic compounds) were characterized after isolation by hydrodistillation and analysis by GC and GC-MS. The seeds volatiles were dominated by linalool (82%), with γ-terpinene (4%), camphor (3%) and geraniol (3%) as other compounds. Linalool was present in the volatile fractions of clones A and B in vitro samples and of in vivo aerial parts, always in small relative amounts (0.1%, 0.1% and 0.3%, respectively). Dodecanal (17%), dodecanol (17%), n-tetradecanol (15%) and decanal (10%) were the dominant volatiles in the in vivo vegetative parts. β-Phellandrene (37% in A, 45% in B), terpinolene (9% in both), β-sesquiphellandrene (4% in A, 6% in B) and α-phellandrene (2% in A, 3% in B) were the major identified volatiles in clones A and B in vitro samples. Despite the purple coloration in clone A plants, the volatile profile was quantitative and qualitatively very similar to clone B. The volatiles composition of in vitro grown plants was qualitatively similar to the seeds volatiles and very different from the in vivo aerial parts volatiles. Lipophilic (tocopherols, carotenoids and chlorophylls) and hydrophilic (sugars, ascorbic acid, phenolics, flavonols and anthocyanins) compounds were quantified. Furthermore, the antioxidant properties were evaluated by free radical scavenging activity, reducing power and lipid peroxidation inhibition. In vivo aerial parts showed the highest antioxidant activity mainly due to its highest levels of hydrophilic compounds. Otherwise, in vitro samples, mainly clone A, gave the highest concentration in lipophilic compounds but a different profile when compared to the in vivo vegetative parts. Clones A and B revealed a lack of β -carotene, β- and δ-tocopherols, a decrease in α -tocopherol, and an increase in ү -tocopherol, and an increase in y-tocopherol and chlorophylls in comparison to the in vivo sample. A detailed analysis of individual phenolic compounds present in in vivo and in vitro grown samples, was also performed. In vivo aerial parts showed quercetin derivatives as the main flavonoids, and quercetin-3-O-rutinoside was the main polyphenol found in this part of coriander (3296 mg/kg dw). The seeds revealed only phenolic acids and derivatives, being caffeoyl N-tryptophan hexoside the most abundant phenolic derivative (45.33 mg/kg dw). In vitro samples also gave a high diversity of polyphenols, being C-glycosylated apigenin the main compound (2983 mg/kg dw). Anthocyanins were only found in clone A, which was certainly related to its purple pigmentation, and peonidin-3-O-feruloylglucoside-5-O-glucoside was the major anthocyanin found (1.70 μg/kg dw). With molecular analysis we could distinguish in vitro grown individuals of clone A and clone B based on phenotypic differences, using in vivo aerial parts individuals as an out group control. The ISSR cluster showed no correlation between in vivo and in vitro grown samples (similarity index below 0.30). In vitro individuals revealed a high similarity index (0.70), meaning that the genetic variation is low and could not explain the phenotypic differences found between the two clones. In vitro culture might be useful to explore the plants potentialities for industrial applications, controlling environmental conditions to produce higher amounts of some bioactive products. It can also be used to explore new industrial, pharmaceutical and medicinal potentialities, such as the production of secondary metabolites like volatiles and phenolic compounds.
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Citation
Dias, Maria Inês Moreira Figueiredo (2011). Caracterização química e molecular de amostras de Coriandrum sativum L. obtidas in vivo e in vitro. Bragança: Escola Superior Agrária. Dissertação de Mestrado em Biotecnologia
Publisher
Instituto Politécnico de Bragança, Escola Superior Agrária