Comparison of methyl eugenol levels and eugenol O-methyltransferase gene structure in different Ocimum plant species

Ocimum tenuiflorum L. (Holy basil or Tulsi), belonging to Lamiaceae family, is an important medicinal plant, with particular religious significance to the Hindu community throughout the world. Tulsi plants are characterised by high levels of essential oils, containing phenylpropanoids, such as eugenol, methyl eugenol (ME), chavicol and estragole (methyl chavicol). Two chemotypes of Tulsi have been distinguished, based on high or low methyl eugenol:eugenol ratios. As methyl eugenol and methyl chavicol are classed as genotoxic carcinogens, it is important to ensure that the levels of these compounds in herbal products fall below the regulatory thresholds. The levels of methyl eugenol in O. tenuiflorum are generally higher than in other Ocimum species. The conversion of eugenol to methyl eugenol is catalysed by eugenol O-methyltransferase (EOMT) enzyme. EOMT gene sequences have been isolated from a range of Ocimum species and from different chemotypes of O. tenuiflorum. Analyses of EOMT genomic and cDNA sequences revealed a 843 bp open reading frame and the presence of a 90-104 bp intron. Alignment of the protein sequences from several Ocimum species indicates a number of amino acid substitutions that may be correlated with methyl eugenol content and reflect differences in enzyme activity and substrate specificity. Later, the levels of ME along with other three secondary metabolites - eugenol, chavicol and estragole have been determined by HPLC. Full length EOMT cDNA were prepared from O. tenuiflorum, O. gratissimum and O. basilicum plants and are being used to study the properties of the plant EOMT enzyme after expression in bacteria. Along with protein study, the Gateway cloning system was successfully used to prepare four binary vectors [O. basilicum (EOMT), O. basilicum (CVOMT), O. tenuiflorum and O. gratissimum] for future plant transformation experiments

DNA Authentication of St John’s Wort (Hypericum perforatum L.) Commercial Products Targeting the ITS Region

open access articleThere is considerable potential for the use of DNA barcoding methods to authenticate raw medicinal plant materials, but their application to testing commercial products has been controversial. A simple PCR test targeting species-specific sequences within the nuclear ribosomal internal transcribed spacer (ITS) region was adapted to screen commercial products for the presence of Hypericum perforatum L. material. DNA differing widely in amount and extent of fragmentation was detected in a number of product types. Two assays were designed to further analyse this DNA using a curated database of selected Hypericum ITS sequences: A qPCR assay based on a species-specific primer pair spanning the ITS1 and ITS2 regions, using synthetic DNA reference standards for DNA quantitation and a Next Generation Sequencing (NGS) assay separately targeting the ITS1 and ITS2 regions. The ability of the assays to detect H. perforatum DNA sequences in processed medicines was investigated. Out of twenty different matrices tested, both assays detected H. perforatum DNA in five samples with more than 103 ITS copies µL−1 DNA extract, whilst the qPCR assay was also able to detect lower levels of DNA in two further samples. The NGS assay confirmed that H. perforatum was the major species in all five positive samples, though trace contaminants were also detected

Sequence-Specific Detection of Aristolochia DNA – A Simple Test for Contamination of Herbal Products

open access articleHerbal medicines are used globally for their health benefits as an alternative therapy method to modern medicines. The market for herbal products has increased rapidly over the last few decades, but this has in turn increased the opportunities for malpractices such as contamination or substitution of products with alternative plant species. In the 1990s, a series of severe renal disease cases were reported in Belgium associated with weight loss treatment, in which the active species Stephania tetrandra was found to be substituted with Aristolochia fangchi. A. fangchi contains toxic aristolochic acids, which have been linked to kidney failure, as well as cancers of the urinary tract. Because of these known toxicities, herbal medicines containing these compounds, or potentially contaminated by these plants, have been restricted or banned in some countries, but they are still available via the internet and in alternate formulations. In this study, a DNA based method based on quantitative real-time PCR (qPCR) was tested to detect and distinguish Aristolochia subg. Siphisia (Duch.) O.C.Schmidt species from a range of medicinal plants that could potentially be contaminated with Aristolochia material. Specific primers were designed to confirm that Aristolochia subg. Siphisia can be detected, even in small amounts, if it is present in the products, fulfilling the aim of offering a simple, cheaper and faster solution than the chemical methods. A synthetic gBlock template containing the primer sequences was used as a reference standard to calibrate the qPCR assay and to estimate the copy number of a target gene per sample. Generic primers covering the conserved 5.8S rRNA coding region were used as internal control to verify DNA quality and also as a reference gene for relative quantitation. To cope with potentially degraded DNA, all qPCR primer sets were designed to generate PCR products of under 100 bp allowing detection and quantification of A. fangchi gBlock even when mixed with S. tetrandra gBlock in different ratios. All proportions of Aristolochia, from 100 to 2%, were detected. Using standards, associating the copy number to each start quantity, the detection limit was calculated and set to about 50 copies

Qualitative and Qualitative Phytochemical Screening of Medicinal Herbal Ocimum Tulsi Leaves

Department of Pharmacy; Chemistry For Health, De Montfort UniversityThe drastic changes in human life style and food habits over the last century have led to various chronic diseases. Diabetes milletus is one such disease, which is causing serious problems to human health in all parts of the world. Diabetes Mellitus is a metabolic disorder characterized by high blood glucose levels due to defect in insulin secretion, insulin action or both. Poor control of the condition can result in long term complication such as diabetic nephropathy, neuropathy, retinopathy, hypertension, dyslipidaemia and cardiovascular disease, which can lead to a heart attack or stroke. Scientific reports revealed that diabetes cannot be cured completely. Current therapy for diabetes is centred around controlling blood sugar levels to keep them in the normal range. However, diabetes is often associated with dyslipidaemia and oxidative stress, which contributes to the damage caused to organs. Therefore, to adequately control diabetes, blood lipid levels and oxidative stress need to be controlled alongside blood glucose. The modern systems of medicine have not shown any significant effect for the treatment of the disease. There are also concerns about the cost and safety in long-term use for some of these 5th Annual International Conference on Health & Medical Sciences, 1-4 May 2017, Athens, Greece: Abstract Book 136 drugs. Hence focus has been turned towards traditional system of medicine. Medicinal plants play an important role in management of diabetes milletus. During the past few years many bioactive drugs have been isolated from plants. Herbal medicines have shown good clinical practice in the therapy of diabetic milletus. The genus Ocimum is of the basil family Lamiaceae, which contains approximately 150 species of aromatic plants that are found mainly in the tropical regions of the world. Many of these species have extensive medicinal applications in indigenous medicinal systems of countries in Asia, Africa and South America. Lamiaceae is one of the holiest and sacred herbs grown in India. This plant is known to possess antiseptic, analgesic, anti-inflammatory, antimicrobial, antistress, Immunomodulatory, hypoglycaemic, hypotensive and antioxidant properties. Among these species are Ocimum sanctum L. or Ocimum tenuiflorum L., also known as Tulsi or Holybasil, an aromatic plant. There are two sub- types of Ocimum sanctum called Krishna tulsi which is the black-purple variety, and the white Vana tulsi. It is widely used in Ayurveda and Siddha system of medicine to cure various ailments. The dried powder of Tulsi was placed in the thimble of Soxhlet apparatus, distilled water employed as a solvent. The extract was concentrated using Rotavapor. Then the extract was dried in a digital water bath till a dark green residue was obtained. The percentage yield was 10%w/w. The test residue and powder was subjected to phytochemical analysis to find out the presence of phytochemical constituents and compared. The phytochemical tests employed for alkaloids and tannins, Cardiac glycosides, saponins, terpenoids and flavonoids. The latter are known for their strong antioxidant properties. Here reported investigation carried out to determine the qualitative and qualitative analysis of phytochemical screening and possible chemical components including assessment of antioxidant properties. The analytical techniques employed for chemical investigation comprised Atomic Absorption Spectroscopy, Thin Layer Chromatography, Mass Spectroscopy, Infrared spectrophotometry, UV and Visible spectroscopy, High Pressure Liquid Chromatography, Nuclear Magnetic Resonance.and scanning electron microscope. Ocimum sanctum L. leaves analysis of hydro alcoholic extract revealed mainly Eugenol, an important phytochemical bioactive compound of Ayurvedic and other marketed herbal formulations. This study is currently in progress