Phytochemical Diversity of Murraya koenigii (L.) Spreng. from Western Himalaya

Murraya koenigii (L.) Spreng. (Rutaceae), commonly known as ‘curry leaf tree’, is a popular spice and condiment of India. To explore the diversity of the essential‐oil yield and aroma profile of curry leaf, growing wild in foot and mid hills of north India, 58 populations were collected during spring season. M. koenigii populations were found to grow up to an altitude of 1487 m in north India. Comparative results showed considerable variations in the essential‐oil yield and composition. The essential‐oil yield varied from 0.14 to 0.80% in shade‐dried leaves of different populations of M. koenigii. Analysis of the essential oils by GC and GC/MS, and the subsequent classification by statistical analysis resulted in four clusters with significant variations in their terpenoid composition. Major components of the essential oils of investigated populations were α‐pinene ( 2 ; 4.5–71.5%), sabinene ( 3 ; <0.05–66.1%), (E)‐caryophyllene ( 11 ; 1.6–18.0%), β‐pinene ( 4 ; <0.05–13.6%), terpinen‐4‐ol ( 9 ; 0.0–8.4%), γ‐terpinene ( 8 ; 0.2–7.4%), limonene ( 7 ; 1.1–5.5%), α‐terpinene ( 6 ; 0.0–4.5%), (E)‐nerolidol ( 14 ; 0.0–4.1%), α‐humulene ( 12 ; 0.6–3.5%), α‐thujene ( 1 ; 0.0–2.5%), β‐elemene ( 10 ; 0.2–2.4%), β‐selinene ( 13 ; 0.2–2.3%), and myrcene ( 5 ; 0.5–2.1%). Comparison of the present results with those in earlier reports revealed new chemotypes of M. koenigii in investigated populations from Western Himalaya. The present study documents M. koenigii populations having higher amounts of sabinene ( 3 ; up to 66.1%) for the first time.     

Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a^−/−, Epm2b^−/−, and Epm2a^−/− Epm2b^−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b^−/− and Epm2a^−/− Epm2b^−/− cells) but not laforin (Epm2a^−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.     

Enhancement of taxol production from endophytic fungus Fusarium redolens

The optimization of taxol production by Fusarium redolens by one factor at a time (OFAT) approach led to production of 70 μg/L of taxol. With sucrose and NH₄NO₃ as the carbon and nitrogen sources and medium volume (V _m ) to flask volume (V _f ) ratio of 0.2, a greater taxol production was attained. NH₄NO₃, MgSO₄?7H₂O and NaOAc at 6.25, 0.63, and 1.25 g/L, were the significant factors for attaining the highest taxol production. The optimization of culture variables led to the production of taxol from 66 to 198 μg/L, which is three fold higher than that in the unoptimized medium. Current study results suggested the success of Response Surface Methodology in enhancing the production of fungal taxol.     

Molecular Profiling for Genetic Variability in <Emphasis Type="Italic">Capsicum</Emphasis> Species Based on ISSR and RAPD Markers

The taxonomic identity of Capsicum species is found to be difficult as it displays variations at morpho-chemical characters. Twenty-two accessions of six Capsicum species, namely, C. annuum, C. baccatum, C. chinense, C. eximium, C. frutescens, and C. luteum were investigated for phenotypic diversity based on flower color and for genetic differences by molecular makers. The genetic cluster analyses of 27 RAPD and eight ISSR primers, respectively, revealed genetic similarities in the ranges of 23–88% and 11–96%. Principal component analysis of the pooled RAPD and ISSR data further supports the genetic similarity and groupings. Different species showed variations in relation to corolla shade of flower. C. annuum accessions formed a single cluster in the molecular analysis as maintaining their flower characteristic. C. chinense accession shared flower features with the accessions of C. frutescens and were found to be closer at genotypic level. C. luteum was found to be rather closer to C. baccatum complex, both phenotypically and genetically. The only accession of C. eximium presenting purple flowers falls apart from the groupings. The floral characteristics and the molecular markers are found to be useful toward the delineation of the species specificity in Capsicum collection and identification of genetic stock.     

Genetic and Chemical Diversity of High Mucilaginous Plants of <Emphasis Type="Italic">Sida</Emphasis> Complex by ISSR Markers and Chemical Fingerprinting

A method was developed based on multiple approaches wherein DNA and chemical analysis was carried out toward differentiation of important species of Sida complex that is being used for commercial preparation. Isolated DNA samples were successfully performed through PCR amplification using ISSR markers and degree of genetic diversity among the different species of Sida is compared with that of chemical diversity. For genetic fingerprint investigation, selected 10 ISSR primers generating reproducible banding patterns were used. Among the total of 63 amplicons, 62 were recorded as polymorphic, genetic similarity index deduced from ISSR profiles ranged from 12 to 51%. Based on similarity index, S. acuta and S. rhombifolia found to be most similar (51%). High number of species-specific bands played pivotal role to delineate species at genetic level. Investigation based on HPTLC fingerprints analysis revealed 23 bands representing to characteristic chemicals and similarity index ranged from 73 to 91%. Prominent distinguishable bands were observed only in S. acuta, while S. cordifolia and S. rhombifolia shared most bands making them difficult to identify on chemical fingerprint basis. This report summarizes the genotypic and chemotypic diversity and the use of profiles for authentication of species of Sida complex.     

Impact of Geographic Range on Genetic and Chemical Diversity of Indian Valerian (<Emphasis Type="Italic">Valeriana jatamansi</Emphasis>) from Northwestern Himalaya

An effort was made to determine the impact of geographic range on genetic richness and chemical constituents of Valeriana jatamansi Jones, an herb indigenous to the northwestern Himalaya. The genetic structure of 16 accessions from two major divisions of Uttarakhand state (Kumaon and Garhwal) was analyzed by ISSR markers. Overall genetic diversity among the populations was 45 %, with a cumulative range of 35–92 % similarity for most of the high-altitude plants and a comparatively narrow range, 50–88 %, for the population below the altitude of 1,800 m. Likewise, a remarkable predictability was evident from the chemical constituents on an individual basis. In principal component analysis, most of the accessions fall into two major groups and are classified as chemotypes based on the percentage of similar chemical constituents; these are mostly correlated to altitude. Geographic distance seems to influence the genetic and chemical variability, indicating the genetic inbreeding within the population.     

Diversity and antimitotic activity of taxol-producing endophytic fungi isolated from Himalayan yew

Endophytic fungi represent an under explored resource of novel lead compounds and have the capacity to produce diverse classes of plant secondary metabolites. Here, we investigated the endophytic fungal diversity of taxol-producing endophytes from Taxus baccata L. ssp. wallichiana (Zucc.) Pilger and also tested the antimitogenic effect of fungal taxol using potato disc tumor assay. A total of 60 fungal endophytes were isolated from the inner bark (phloem-cambium) of T. baccata ssp. wallichiana, collected from different locations of the northern Himalayan region. Two key genes, DBAT (10-deacetylbaccatin III-10-O-acetyl transferase) and BAPT (C-13 phenylpropanoid side chain-CoA acyltransferase), involved in taxol biosynthesis were used as molecular markers for the screening of taxol-producing strains. Five representative species gave positive amplification hits by molecular marker screening with the bapt gene. These fungi were characterized and identified based on morphological and molecular identification. The taxol-producing capability of these endophytic fungi was validated by HPLC-MS. Among the five taxol-producing fungi, the highest yield of taxol was found to be 66.25 μg/l by Fusarium redolens compared with those of the other four strains.