ABSTRACT
Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.
Subject(s)
Saccharomyces cerevisiae/metabolism , Sesquiterpenes/metabolism , Pogostemon , Oils, Volatile/metabolismABSTRACT
Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.
Subject(s)
Amino Acid Sequence , Cloning, Molecular , Geranyltranstransferase/genetics , Pogostemon , Transcription Factors/geneticsABSTRACT
Background: the current research studies why it is effective using Anrederacordifolia, Psidium guajava, and Pogostemon cablin by the local community as a traditional medicine for diarrhea treatment caused by Escherichiacoli bacteria. Objectives: We compared the inhibitor effectiveness of three leaf extracts against Escherichia coli; we also identified the anti-bacterial substances contained in leaf extracts. Methods: We determined the bacterial test activity using the "agar diffusion" method and the thin layer chromatography (TLC) as qualitative analysis for determining the anti-bacterial substances contained in the extract. Results: The Pogostemon cablin leaf extract contained terpenoids, phenolic, and flavonoids compound as bacterial inhibitors, and the comparison showed that Pogostemon cablin leaf extract had the greatest bacterial inhibition power. Conclusion: The antibiotic substances found in the leaf extracts of Anredera cordifolia, Psidium guajava, and Pogostemon cablin can be used as traditional medicine. The breakthrough was evidenced by the ability to inhibit Escherichia coli bacteria. This research shows that traditional medicine has ancient knowledge used by this paper
Antecedentes: la presente investigación estudia la eficacia del uso de Anredera cordifolia, Psidium guajava y Pogostemoncablin por la comunidad local como medicina tradicional para el tratamiento de la diarrea causada por la bacteria Escherichia coli. Objetivos: Comparamos la eficacia inhibidora de los extractos de tres hojas contra Escherichia coli; también identificamos las sustancias antibacterianas contenidas en los extractos de hojas. Métodos: Determinamos la actividad de la prueba bacteriana mediante el método de "difusión en agar" y la cromatografía en capa fina (TLC) como análisis cualitativo para determinar las sustancias antibacterianas contenidas en el extracto. Resultados: el extracto de hoja de Pogostemoncablin contenía compuestos terpenoides, fenólicos y flavonoides como inhibidores bacterianos, y la comparación mostró que el extracto de hoja de Pogostemon cablin tenía el mayor poder de inhibición bacteriana. Conclusión: El contenido de sustancias antibióticas que se encuentran en el extracto de hoja de Anredera cordifolia, Psidium guajava y Pogostemoncablin puede utilizarse como medicina tradicional. Esto se evidencia por la capacidad de inhibir la bacteria Escherichiacoli. Esta investigación muestra que la medicina tradicional tiene un conocimiento antiguo utilizado por este artículo
Subject(s)
Humans , Anti-Bacterial Agents , Agar , Psidium , Escherichia coli , Pogostemon , Anti-Infective AgentsABSTRACT
This study aims to explore the relationship of DNA methylation with the contents of the index components as well as the growth and development of Pogostemon cablin. The demethylation reagent 5-azacytidine(5-azaC) was used to treat the tissue culture seedlings of patchouliol-type P. cablin. High performance liquid chromatography was employed to evaluate the changes of DNA methy-lation in P. cablin, and GC-MS to detect the contents of index components in P.cablin. The agronomic characters of P.cablin were measured using the common methods. The results showcased that DNA methylation of P.cablin was significantly reduced by 5-azaC in a concentration-dependent manner. Thirty days after treatment with 5-azaC at different concentrations, the content of patchouli alcohol changed slightly; compared with that in the control group, the content of pogostone in 50 μmol·L~(-1) and 100 μmol·L~(-1) 5-azaC groups was significantly up-regulated. The 100 μmol·L~(-1) 5-azaC group had the largest differences in contents of pogostone and patchouli alcohol compared with the control group, followed by the 50 μmol·L~(-1) 5-azaC group. Ninety days after disinhibition, the content of pogostone in the treatment group was significantly increased and the content of patchouli alcohol was significantly decreased. In addition, 5-azaC significantly inhibited the growth and development of P.cablin in a dose-dependent manner. These results indicate that DNA methylation regulates the biosynthesis of the index components in patchouliol-type P.cablin and proper demethylation can directly promote the synthesis of pogostone and indirectly affect the accumulation of patchouli alcohol.
Subject(s)
Azacitidine , DNA Methylation , Gas Chromatography-Mass Spectrometry , Oils, Volatile , Pogostemon/geneticsABSTRACT
The terpenoids in Pogostemon cablin have complex structures and abundant pharmacological effects. Patchouli alcohol(PA) and pogostone(PO) have a high medicinal value by virtue of anti-tumor, anti-inflammatory, antibacterial, antioxidant, and other biological activities. Due to the low content of terpenoid metabolites in P. cablin, the study of biosynthesis and metabolism regulation can provide a biosynthetic basis for obtaining high-content terpenoids. In this study, key enzyme genes in biosynthesis, transcription factors in metabolism regulation, spatio-temporal expression of terpene synthase were reviewed, aiming to provide a reference for the development, protection, and utilization of P. cablin resources.
Subject(s)
Pogostemon/genetics , Terpenes , Transcription Factors/geneticsABSTRACT
Patchouli [Pogostemon cablin (Blanco) Benth.] is a plant of the family Lamiaceae, widely used as an essential oil in the cosmetics and perfumery industry. This study aimed to evaluate the influence of storage time and temperature of dry leaves on the patchouli essential oil content and chemical composition. The experiment was performed in a completely randomized design in a 6x2x2 factorial scheme, testing storage time (0, 1, 2, 4, 8, and 16 weeks) and temperature (28°C and 33°C) of dry leaves of two patchouli genotypes (POG-015 and POG-021). The variables essential oil content and chemical composition, and the identification of fungus during storage were evaluated. Results showed that the storage significantly influenced the essential oil content. Patchoulol was identified as the major compound in both genotypes, ranging from 55.05% to 68.77% (POG-15) and from 52.83% to 64.06% (POG-021). Based on the results of patchoulol, dry leaves of both genotypes (POG-015 and POG-021) can be stored for up to eight weeks at 28- 33°C without altering the essential oil quality.
Patchouli [Pogostemon cablin (Blanco) Benth.] é uma planta pertencente à família Lamiaceae, e seu óleo essencial é utilizado nas indústrias de perfumes e cosméticos. O objetivo do trabalho foi avaliar a influência do tempo e temperaturas de armazenamento das folhas secas no teor e na composição química do óleo essencial de patchouli. O delineamento experimental foi o inteiramente casualizado em esquema fatorial 6x2x2, testando tempo de armazenamento (0, 1, 2, 4, 8 e 16 semanas), temperatura de armazenamento (28°C e 33°C) de folhas secas de dois genótipos (POG-015 e POG-021) de patchouli. As variáveis avaliadas foram o teor e a composição química do óleo essencial e a identificação dos fungos que se desenvolveram durante o armazenamento. Os resultados mostraram que o armazenamento influenciou significativamente o teor de óleo essencial. Dos compostos identificados, o patchoulol foi o composto majoritário nos dois genótipos, variando de 55,05% a 68,77% (POG-15) e 52,83% a 64,06% (POG-021). Baseado no patchoulol, folhas secas dos genótipos (POG-015 e POG-021) de patchouli podem ser armazenadas por um período de até oito semanas em temperatura de 28 e 33°C, sem alterar a qualidade do óleo essencial.
Subject(s)
Oils, Volatile , PogostemonABSTRACT
Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.
Subject(s)
DNA Transposable Elements , Electroporation , Genes, Bacterial , Mutagenesis, Insertional , Pogostemon , Microbiology , Ralstonia solanacearum , Genetics , VirulenceABSTRACT
Background: Defense-related anti-oxidative response is a vital defense mechanism of plants against pathogen invasion. Ralstonia solanacearum is an important phytopathogen. Bacterial wilt caused by R. solanacearum is the most destructive disease and causes severe losses in patchouli, an important aromatic and medicinal plant in Southeast Asia. The present study evaluated the defense response of patchouli inoculated with virulent R. solanacearum. Results: Results showed that the basic enzymatic activities differed not only between the leaves and stems but also between the upper and lower parts of the same organ of patchouli. POD, SOD, PPO, and PAL enzymatic activities were significantly elevated in leaves and stems from patchouli inoculated with R. solanacearum compared to those in control. The variation magnitude and rate of POD, PPO, and PAL activities were more obvious than those of SOD in patchouli inoculated with R. solanacearum. PAGE isoenzymatic analysis showed that there were one new POD band and two new SOD bands elicited, and at least two isoformic POD bands and two SOD bands were observably intensified compared to the corresponding control. Conclusion: Our results suggest that not only defense-related enzymatic activities were elevated but also the new isoenzymatic isoforms were induced in patchouli inoculated with R. solanacearum.
Subject(s)
Ralstonia solanacearum/pathogenicity , Pogostemon/enzymology , Pogostemon/microbiology , Phenylalanine Ammonia-Lyase/metabolism , Superoxide Dismutase/metabolism , Virulence , Catechol Oxidase/metabolism , Peroxidase/metabolism , Ralstonia solanacearum/physiology , Electrophoresis, Polyacrylamide Gel , Enzymes/immunology , Enzymes/metabolism , Native Polyacrylamide Gel Electrophoresis , Pogostemon/immunology , AntioxidantsABSTRACT
RESUMOPatchouli é uma espécie aromática pertencente à família Lamiaceae que possui óleo essencial com alto valor comercial devido à aplicação nas indústrias de higiene, cosméticos e perfumarias. Considerando as características voláteis e estruturas de armazenamento em diferentes órgãos das plantas aromáticas, a determinação de métodos de secagem é de extrema importância e pode afetar tanto o rendimento como a composição do óleo essencial. Este trabalho teve como objetivo principal avaliar o teor e a composição do óleo essencial de patchouli após secagem por diferentes períodos em estufa e a temperatura ambiente. O experimento foi realizado durante o mês de fevereiro de 2011 em delineamento experimental inteiramente casualizado em esquema fatorial 2X6, correspondente a dois métodos de secagem (estufa 65ºC e temperatura ambiente) e seis tempos para o início da extração do óleo essencial após a (0, 2, 4,6,8 e 10 dias após a colheita),com três repetições. O óleo essencial foi extraído por meio hidrodestilação em aparelho graduado de Clevenger e os constituintes do óleo essencial foram analisados por cromatografia em fase gasosa acoplada à espectrometria de massas. Houve interação significativa entre os métodos e tempos de secagem tanto para o teor como para a composição do óleo essencial. A secagem das plantas por dois dias em estufa a 65ºC aumentou o teor de óleo essencial, após este período houve redução significativa. Para as folhas secas em temperatura ambiente, o teor de óleo essencial foi superior aos quatro e aos seis dias após a colheita. Comparando-se os métodos, a secagem em estufa mostrou-se mais eficiente, sendo o teor significativamente superior após dois dias de secagem. Após este período, no entanto, houve drástica redução do teor de óleo essencial, sendo inferior ao teor observado em todos os períodos nas folhas secas em temperatura ambiente. Os diferentes métodos e tempos de secagem alteraram também a composição do óleo essencial. Embora a secagem em estufa a 65º por dois dias não tenha resultado em redução dos teores dos constituintes majoritários patchoulol e pogostol, maiores teores foram observados após seis ou oito dias de secagem. Quando a secagem foi realizada a temperatura ambiente, não houve alteração dos teores de ambos os constituintes.
ABSTRACTPatchouli is an aromatic species belonging to the Lamiaceae family that has essential oil with high commercial value to the hygiene, cosmetic and perfume industries. Considering its volatile characteristics and storage structures in different organs of aromatic plants, the determination of drying methods is extremely important and can affect both the yield and the composition of the essential oil. This work aimed to evaluate the content and composition of the patchouli essential oil after drying it in different periodin an oven and at room temperature. The trial was carried out in February 2011 at an experimental design completely randomized in 2X6 factorial scheme, corresponding to two drying methods (oven at 65˚C and room temperature) and six periods of essential oil extraction after harvest (0, 2, 4, 6, 8, and 10 days after harvest), with three replications. The essential oil was extracted by hydrodistillation in a Clevenger apparatus and the essential oil components were analyzed by gas chromatography coupled to mass spectrometry. There was significant interaction among the methods and drying times for both the content as well as for the composition of essential oil. The drying of the plants for two days in an oven at 65˚C increased the essential oil content; after this period, there was a significant reduction. For the leaves that dried at room temperature, their contentof essential oil was superior at the fourth and sixth days after the harvest. When comparing the methods, the drying in the oven was more efficient, with the oil content being significantly higher after two days of drying. After this period, however, there was a drastic reduction in the essential oil content, lower than the one observed in all periods when the leaves dried at room temperature. The different methods and drying periods also altered the essential oil`s composition. Although drying in oven at 65˚ for two days did not result in reduction of the contents of patchoulol and pogostol, higher contents were observed after six or eight days of drying. When drying was carried out at room temperature, no change in relative content of both components was observed.
Subject(s)
Temperature , Oils, Volatile/metabolism , Greenhouses/classification , Pogostemon , Secondary MetabolismABSTRACT
Neste trabalho avaliou-se o efeito da aplicação no plantio e na manutenção de diferentes doses de nitrogênio (30, 60, 90 e 120 Kg ha-1) no desenvolvimento do patchouli, bem como no rendimento e composição do óleo essencial. O experimento foi conduzido no município de Joinville-SC, de agosto de 2006 a maio de 2007, com delineamento em blocos ao acaso em parcelas subdivididas no tempo, com cinco repetições. Foram executados duas épocas de adubação para avaliação dos níveis de nitrogênio, após o plantio e após primeiro corte, na manutenção. Avaliouse o acúmulo de massa seca foliar e de ramo, número e comprimento de ramos, área foliar da planta e rendimento e composição do óleo essencial. A aplicação de 98 Kg ha-1 de nitrogênio na manutenção proporcionaram maiores ganhos de massa seca foliar. Maior rendimento de óleo essencial foi obtido no segundo corte, após a adubação de manutenção. A composição do óleo essencial, aparentemente, não sofreu influência dos diferentes níveis de nitrogênio aplicados em ambas as épocas (plantio e manutenção). As médias do constituinte majoritário patchoulol variaram entre 45,66% e 49,54%.
This work evaluated the effect of the application in the planting and maintenance of different levels of nitrogen fertilization (30, 60, 90 and 120 Kg ha-1) in the development of patchouli. The experiment was carried out at Joinville-SC from August 2006 the May 2007. The experimental design was completely randomized blocks subdivided in time, with five replications. Two time fertilization were carried out to evaluate the levels of nitrogen, after planting and after first cut, in maintenance. It was determined the leaf and branch dry mass, number and length of branches, leaf area and essential oil yield and composition. The application of 98 Kg ha-1 of nitrogen in maintenance provided greater gains of leaf dry mass. Largest essential oil yield was obtained in the second cut, after fertilization. The composition of the essential oil was not affected by different nitrogen levels in both the times (plantation and maintenance). The averages of the main constituent patchoulol varied from 45,66% to 49.54%.
Subject(s)
Oils, Volatile , Crop Production , Pogostemon , NitrogenABSTRACT
O patchouli possui óleo essencial nas folhas com utilização principalmente na indústria de perfumaria. O objetivo foi avaliar o melhor tempo de extração de óleo essencial de folhas secas de patchouli. Os tratamentos foram 1, 2, 3, 4, 5, 6, 7 e 8 horas de extração, através do método de hidrodestilação, com aparelho graduado do tipo Clevenger e balões com capacidade de 2 L.O delineamento foi inteiramente casualizado com três repetições. O material destilado foi seco à sombra até atingir aproximadamente 20% de umidade. Para cada tratamento, foram utilizadas amostras de 50 g de massa seca foliar. Foram avaliados o rendimento e a composição do óleo essencial. Não houve diferença entre os diferentes tempos de extração no rendimento de óleo essencial, podendo a extração do óleo essencial de patchouli ser realizada com uma hora de extração. O tempo de extração aumenta as porcentagens relativas do beta-guaieno (0,81%), beta-patchouleno (1,26%), alfa-selineno (1,37%), cariofileno (2,44%), alfa-patchouleno (3,08%) e gama-patchouleno (4,82%). O teor de pogostol (5,11%) reduz com o aumento do tempo de extração. O patchoulol, alfa-guaieno, alfa-bulneseno e seicheleno não sofrem influencia do tempo de extração.
Patchouli accumulatea an essential oil on its leaves, and it is mainly used by the perfume industry. The purpose of this work was to determine the distillation time for the extraction of essential oils from the leaves of patchouli. The treatments included 1, 2, 3, 4, 5, 6, 7 and 8 hours of extraction through the hydrodistillation method, using a Clevenger apparatus. The experimental design was completely randomized with three replications. The leaves were dried at room temperature until they reached 20% of humidity. The essential oil yield was measured from samples containing 50g of leaf dry mass. The essential oil production and composition were evaluated. No differences among the treatments were found on the essential oil yield, suggesting that the essential oil extraction can be carried out for 1 hour according to the experimental conditions. However, extending the extraction time, an increase in the relative percentage of beta-patchoulene (1.26%), cariofilene (2.44%), gamma-patchoulene (4.82%), alpha-patchoulene (3.08%), beta-guaiene (0.81%) and alpha-selinene (1.37%) was observed. The pogostol (5.11%) content was reduced with the increase of the extraction time. Patchoulol, alpha-guaieno, alpha-bulneseno and seicheleno contents were not affected by the evaluated extraction times.
Subject(s)
Oils, Volatile/analysis , Plant Leaves , Pogostemon/classification , Distillation/methods , LiftingABSTRACT
Pogostemon cablin (Blanco) Benth., comumente conhecido como patchouli, é uma espécie aromática utilizada pela indústria de perfumes devido a fragrância amadeirada e propriedade fixadora do óleo essencial. Fatores que afetam o teor dos constituintes do óleo essencial devem ser avaliados visando obter matéria prima de melhor qualidade. O experimento foi conduzido em casa de vegetação com delineamento inteiramente casualizado em esquema fatorial 2x3 e 4 repetições, sendo dois níveis de giberelina (0 e 200 mg L-1) e três níveis de extrato de alga marinha (0, 15 e 30 mg L-1). A aplicação dos reguladores vegetais foi realizada 30 dias após o plantio das mudas, e a avaliação do experimento deu-se 45 dias após a aplicação dos tratamentos. Não houve diferença significativa para a altura das plantas, a concentração de extrato de alga marinha de 15 mg L-1 promoveu aumento no número de folhas em relação à testemunha e a concentração de 30 mg L-1 promoveu decréscimo. Em relação à área foliar e ao número de folhas houve relação inversa para os níveis de extrato de alga marinha, onde o tratamento com maior concentração do regulador resultou em aumento da área foliar e redução do número de folhas. O teor de óleo essencial foi superior após a aplicação de 15 mg L-1 de extrato alga marinha isoladamente ou combinado com GA3. A produtividade do óleo essencial também aumentou com a aplicação 15 mg L-1 de extrato de alga marinha e quando utilizado somente GA3. A utilização de extrato de alga marinha aumentou a concentração de patchoulol no óleo essencial.
Pogostemon cablin (Blanco) Benth., commonly known as patchouli, is an aromatic species used by the perfume industry due to its woody fragrance and the fixative characteristic of its essential oil. Factors affecting the content of the constituents of essential oils should be evaluated in order to obtain raw materials with better quality. The experiment was carried out in a greenhouse in a completely randomized 2x3 factorial arrangement and four replications, with two levels of gibberellin (0 and 200 mg L-1) and three levels of seaweed extract (0, 15 and 30 mg L-1). Plant regulators were applied thirty days after planting the seedlings; the experiment was evaluated forty-five days after the treatments. There was no significant difference in plant height; the 15 mg L-1 concentration of seaweed extract promoted an increase in the number of leaves in relation to the control; and the 30 mg L-1 concentration promoted a decrease. In relation to leaf area and leaf number, there was an inverse relationship for the seaweed extract levels, in which the treatment with higher a concentration of the regulator resulted in an increased leaf area, reducing the number of leaves. The essential oil content was higher after applying 15 mg L-1 of seaweed extract alone or combined with GA3. The yield of essential oil also increased by applying 15 mg L-1 of seaweed extract and when only GA3 was used. The use of seaweed extract increased the patchoulol concentration in the essential oil.