Chemical and Molecular Characterization of Marigold (Tagetes Species) Found in Pakistan

Abstract

Tagetes, a genus of flowering marigold is a medicinal plant grown over a wide range of climatic conditions. Molecular and chemical characterization of this important plant species may lead to identify novel compounds of high medicinal value. Major goal of the current research project was to characterize Tagetes species on chemical and molecular bases. The specific objectives of the study were to optimize extraction protocols for DNA and chemical compounds, molecular based genetic diversity of Tagetes plants, develop and characterize gene based markers, identify and characterize new/novel compounds present in the species. Fifteen genotypes of Tagetes including hybrids from different parts of Pakistan were collected and used. The plants of all Tagetes were grown in pots and field at CIIT Abbottabad for molecular analyses where as bulk amount of Tagetes minuta was collected and used for chemical analyses. The DNA from all genotypes was isolated using fresh, dried leaf tissues and seeds. Specific primers were developed for limonene synthase gene using sequence analyses and bioinformatic tools. Twenty-five random (RAPD) and 5 specific (degenerate-STS) primers were used for genetic diversity analysis. Chemical compounds were extracted using standard solvent extraction techniques and the crude fractions were tested for biological activities. The fractions were further purified and analyzed by chromatographic (TLC and HPLC) and spectroscopic (LCMS and NMR) techniques for compound identification and structural elucidation. The DNA isolated from fresh leaves and seeds of Tagetes was of good quantity and quality for PCR analyses. Significant variations were observed among all the genotypes of Tagetes species using PCR based markers. Twenty RAPD primers out of 25 and 3 degenerate-STS primers out of 5 revealed high polymorphism rate of 95.21% and 96.55% respectively. The phenograms generated using both marker systems grouped 15 Tagetes genotypes into 4 distinct groups, while T. minuta was placed genetically as most distantly related and unique species. PCR analysis with limonene synthase gene specific degenerate primers amplified candidate gene for limonene synthase in T. minuta. Bioactive extracts from flower parts of Tagetes exhibited antimalarial, phytotoxic, insecticidal and antibacterial activity. The HPLC profile revealed that butanol fraction contained good quality and quantity of flavonoids (17 flavonols) as compared to ethyl acetate (2 falvonols) and chloroform (mixture of other compounds). A total of 19 falvaonols were identified, of which 8 were structurally elucidated. Four new flavonols viz; 6-hydroxy quercetin 7-O-β-(6''-galloylglucopyranoside) (2), 6-hydroxy quercetin 7- O-β-(6''- caffeoylglucopyranoside) (9), 6-hydroxy kaempferol 7-O-β-glucopyranoside (5), 6-hydroxy kaempferol 7-O-β-(6''-galloylglucopyranoside) (7) were identified using butanol fraction. Four known flavonols, viz; 6-hydroxyquercetin 7-O-β-glucopyranoside (1), 6-methoxy quercetin 7-O-β-glucopyranoside (6), 6-methoxyquercetin (19), and 6- hydroxyquercetin (18) were also identified using butanol (1, 7) and ethyl acetate (18, 19) fractions. The combined molecular and biochemical information obtained will lead us to improve Tagetes for highly valuable compounds which may generate foreign exchange by exporting this important medicinal plant to other parts of the world.