Parvaiz H. Qazi

Phylogenetic patterns and genetic diversity of Indian Tinospora species based on chloroplast sequence data and cytochrome P450 polymorphisms

Based on morphology, the species status and taxonomic affinities of three species of Tinospora (T. cordifolia, T. sinensis, and T. crispa) with ranges in India, have been questioned. To evaluate species delimitation and population structure among 40 accessions of the three species, a relatively new marker, cytochrome P450, was used. Five out of nine primers generated polymorphisms with 39 out of 47 bands polymorphic. The P450 binary data, when analyzed using distance methods, strongly supported the monophyly of each Indian species and were congruent with previous RAPD work. To further investigate the status of these species, we combined P450 and RAPD data. The resulting unrooted phylogram highly supports the monophyly of each species but with little population structure within each species. To understand the phylogenetic placement of the three Indian Tinospora species within Menispermaceae, chloroplast atpB and rbcL sequence data for a large sampling of the family were analyzed using likelihood and parsimony methods. The resulting phylogenies highly support the Indian Tinospora species as part of a clade (expanded Tinosporeae), consisting of diverse Menispermaceae from around the world. The three Indian species are monophyletic and are most closely related to Tinospora species from Australia (T. esiangkara and T. smilacina).

Molecular phylogeny in Indian Tinospora species by DNA based molecular markers

The phylogenetic relationships among the three species of Tinospora found in India are poorly understood. Morphology does not fully help to resolve the phylogeny and therefore a fast approach using molecular analysis was explored. Two molecular approaches viz Random Amplified Polymorphic DNA (RAPD) assay and restriction digestion of ITS1-5.8S-ITS2 rDNA (PCR-RFLP) were used to evaluate the genetic similarities between 40 different accessions belonging to three species. Of the 38 random primers used only six generated the polymorphism, while as three out of 11 restriction enzymes used gave polymorphic restriction patterns. The average proportion of polymorphic markers across primers was 95%, however restriction endonucleases showed 92% polymorphism. RAPD alone was found suitable for the species diversions. In contrast PCR- RFLP showed bias in detecting exact species variation. The correlation between the two markers was performed by Jaccards coefficient of similarity. A significant (r= 0.574) but not very high correlation was obtained. Further to authenticate the results obtained by two markers, sequence analysis of ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) was performed. Three independent clones of each species T. cordifolia, T. malabarica and T. crispa were sequenced. Phylogenetic relationship inferred from ITS sequences is in agreement with RAPD data.

A phenylalanine ammonia-lyase ortholog (PkPAL1) from Picrorhiza kurrooa Royle ex. Benth: molecular cloning, promoter analysis and response to biotic and abiotic elicitors.

Picrorhiza kurrooa Royle ex Benth. is a highly reputed medicinal herb utilised in the preparation of a number of herbal drug formulations, principally due to the presence of novel monoterpene iridoid glycosides kenned as picrosides. Phenylalanine ammonia-lyase catalyses an important rate-limiting step in phenylpropanoid pathway and supplies precursors like cinnamic acid, vanillic acid, ferulic acid, etc., to a variety of secondary metabolites including picrosides. The imperilled status of P. kurrooa coupled with lack of information regarding biogenesis of picrosides necessitates deciphering the biosynthetic pathway for picrosides. In the present study, a PAL gene, designated PkPAL1 was isolated from P. kurrooa. The cDNA is 2312 bp in length, consisting of an ORF of 2142 bp encoding for a 713 amino acid protein having a predicted molecular weight of 77.66 kDa and an isoelectric point of pH 6.82. qRT-PCR analysis of various tissues of P. kurrooa showed that PkPAL1 transcript levels were highest in the leaves, consistent with picroside accumulation pattern. Using Genome walking, a 718 bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including TGA-element, TGACG-motif, CGTCA-motif, etc. qRT-PCR indicated up-regulation of PkPAL1 by methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations that corroborated positively with the identified cis-elements within the promoter region. Moreover, altitude was found to have a positive effect on the PkPAL1 transcript levels, driving the expression of PkPAL1 abundantly. Based on docking analysis, we identified eight residues as potentially essential for substrate binding in PkPAL1.

Genetic heterogeneity among keto-acid- producing strains of Gluconobacter oxydans

The genomes of four keto-acid-producing Gluconobacter oxydans strains (ATCC9937, IFO3293, IFO12258 and DSM2343) were analysed by pulse-field gel electrophoresis (PFGE). PFGE of undigested DNA allowed the detection of plasmids in the following strains: ATCC9937 (3 plasmids; 8, 27, 31 kb), IFO3293 (9 kb), DSM2343 (21 kb). The three plasmids in ATCC9937 showed no homology to each other or to plasmids in the other strains. Seventeen restriction enzymes were tested for use in PFGE analysis of the G. oxydans strains and XbaI was chosen for restriction fragment analysis of the genomes. Fairly good resolution of restriction fragments at all size ranges was achieved by using three different pulse–time programs. The genome sizes of the four strains were estimated to be between 2240 kb and 3787 kb. The XbaI restriction patterns of the four strains showed no similarities to each other. Ten random cosmid clones of ATCC9937 were used as hybridization probes against the four strains, but, with the exception of one clone, hybridization signals were only observed with ATCC9937 itself. These data show that the four strains are not closely related.

Isolation, characterization and HPLC quantification of compounds from Aquilegia fragrans Benth: Their in vitro antibacterial activities against bovine mastitis pathogens

ETHNO-PHARMACOLOGICAL RELEVANCE The underground parts of Aquilegia fragrans are traditionally used for the treatment of wounds and various inflammatory diseases like bovine mastitis. However, there are no reports on the phytochemical characterization and antibacterial studies of A. fragrans. AIM OF THE STUDY To isolate compounds from the methanol extract of the underground parts of A. fragrans and determine their antibacterial activity against the pathogens of bovine mastitis. The study was undertaken in order to scientifically validate the traditional use of A. fragrans. MATERIALS AND METHODS Five compounds were isolated from the methanol extract of the underground parts of A. fragrans using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of isolated compounds in the crude methanol extract. The methanol extract and isolated compounds were evaluated for antibacterial activities against mastitis pathogens using broth micro-dilution technique. RESULTS The five isolated compounds were identified as (1) 2, 4-dihydroxyphenylacetic acid methyl ester (2) β-sitosterol (3) Aquilegiolide (4) Glochidionolactone-A and (5) Magnoflorine. A quick and sensitive HPLC method was developed for the first time for qualitative and quantitative determination of four isolated marker compounds from A. fragrans. The crude methanol extract and compound 5 exhibited weak antibacterial activities that varied between the bacterial species (MIC=500-3000 µg/ml). CONCLUSIONS The above results show that the crude methanol extract and isolated compounds from A. fragrans exhibit weak antibacterial activities. Further phytochemical and pharmacological studies are required for proper scientific validation of the folk use of this plant species in the treatment of various inflammatory diseases like bovine mastitis.

ISOLATION, IDENTIFICATION, AND SIMULTANEOUS QUANTIFICATION OF FIVE MAJOR FLAVONOIDS IN EPIMEDIUM ELATUM BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple and reliable HPLC-UV-DAD method was developed for rapid resolution, identification, and quantitation of prenylated flavonoids in Epimedium elatum, a plant endemic to Kashmir Himalayas. Five major prenylated flavonoids namely epimedoside A, epimedin A, epimedin B, epimedin C, and icariin were isolated using repeated column chromatography and simultaneously quantified using HPLC in both underground and aerial parts of E. elatum. All calibration curves showed good linearity (r2 > 0.998) within test ranges and recoveries were 96.8 to 101.2%. The optimized method was successfully applied for the analysis of five major flavonoids in 18 samples of E. elatum collected from three different ecogeographical zones in three different harvesting seasons. The contents of five investigated compounds were greatly variant ranging from 8.73% to 10.96% in aerial parts and 2.52% to 5.06% in underground parts. The concentration of epimedin C was found to be the highest (6.42%) among the five quantified compounds in one of the accessions. The method established in this paper is simple and reliable and could be used for the quality control of E. elatum.

Seasonal Variation in Chemical Composition, Antibacterial and Antioxidant Activities of the Essential Oil of Leaves of Salvia officinalis (Sage) from Kashmir, India

Abstract Salvia officinalis is a versatile medicinal plant with multitude of applications. The present study was designed to study the variation in the quality and quantity of the leaf essential oil of S. officinalis collected during all the months between July 2012 and June 2013, along with its antioxidant and antibacterial activity. The highest percentage of essential oil was found in the month of July (0.600%) and the lowest percentage of oil was recorded in the month of September (0.225%), a period of latent vegetation. The percentage of major constituents of twelve sage samples were: α-pinene (3.22-7.69%), β-pinene (1.57-28.33%), camphene (1.22-6.11%), α-thujone (17.20-40.45%), β-thujone (2.17-4.97%), 1,8-cineole (0.97-7.76%), camphor (0.88-36.97%), borneol (2.12-18.38%), α-caryophyllene (1.38-17.32%) and β-caryophyllene (1.15-15.37 %). The relative percentage of camphor was higher (0.88-36.97%) during cold winters (Nov-Feb). Borneol, αand β-caryophyllene were also at higher percentage in the vegetative period (May-June) while as α-thujone and β-thujone were at peak at the end of vegetative period. The essential oil samples exhibited broad spectrum antibacterial activity against the gram positive bacterial pathogens and showed moderate free radical scavenging activity.

Cytotoxic Potential and Molecular Characterization of Fungal Endophytes from Selected High Value Medicinal Plants of the Kashmir Valley - India.

The present study explores the fungal endophytes from selected high value medicinal plants to check their activities at in-vitro and in-vivo level. The in-vitro cytotoxicity of selected endophytes revealed potent growth inhibition against human cancer cell lines of leukemia (THP-1), lung (A549), prostate (PC-3), colon (Caco-2), neuroblastoma (IMR-32) and breast (MCF-7) at a concentration of 100 µg/ml. Among them the endophytic strains I. e., IIIM2, IIIM3, IIIM7 and IIIM8 showed most significant growth inhibition against colon (Caco-2), prostate (PC-3), lung (A549) and leukemia (THP-1) cancer cell lines. At the in-vivo level maximum (58.95%) tumor growth inhibition was documented with the extract of IIIM2 against Ehrlich Ascites Carcinoma mouse modal. All the potent fungal endophytic strains were characterized using ITS 4 and ITS 5 region sequencing and phylogenetic analysis was ascertained among them. This paper confirms the 2 elite endophytic fungal strains, IIIM2 and IIIM8, have the potential to act as a source of new anticancer compounds.