Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.

Acyltransferases catalyze critical acylation of metabolic intermediates in metabolic pathways and are important in metabolome diversiWcation in secondary metabolism. Terpenes form the largest and most diversiWed group of secondary metabolites (>35,000 compounds). Terpene alcohol acetyltransferases (AATs) are responsible for biogeneration of volatile esters that attribute unique and Wne olfactory aromas to herbs, Xowers, fruits, and foods. AATs catalyze transfer of an acyl group from acetyl/acyl coenzyme A to an alcohol, stoichiometrically generating ester and coenzyme A-SH (CoASH). Recently, the signiWcance of AATs in the Xavor and fragrance of phytogenic foods, drinks, and aromatic oils and in developmental physiology of Xowers and fruits has attracted their biochemical characterization in plants [1–5] for metabolic engineering initiatives. Radioactivity [5] or GC-based quantitation [1,3,5] of ester formed by the reaction are employed to monitor the enzyme activity. Despite their merits, radiometric assays are expensive for several laboratories and have health risks, whereas GC assays are time consuming. Also, the assays estimating ester formation may encounter signiWcant interference due to deacylation of ester formed by ubiquitous esterases. Moreover, for surveying large numbers of samples (for example, screening transgenic populations), use of reliable, cost eVective,

Withania somnifera (Hindi - Ashwagandha, English - winter cherry) is used in Ayurvedic formulations for a variety of health-promoting effects. Several mono- and poly-herbal products commercially available in the Indian market were quantitatively analysed for a number of chemical constituents. The results. revealed wide variations in the content of all seven constituents tested. More than 70-fold variation in the daily intake of withaferin A (the main active constituent of Ashwagandha) was found in the products. The study thus emphasizes the need for stringent phytochemical standardization of herbal products.

ABSTRACT Geranium (Pelargonium graveolens L. Her ex. Aiton) of the Geraniaceae family is a commercially important essential oil plant. The essential oil of the geranium, which has a strong rose-like aroma with minty top note, is extensively used in high-grade perfumery and cosmetic industries. In this investigation, the role of light quality on biosynthesis of the oil (primarily monoterpenoids synthesized and accumulated in leaves) was examined. The third leaf from the top of the plant, previously allowed to photoassimilate 14CO2 in daylight for 1 h, was exposed to blue, green, yellow, orange, red, and far-red colored light. Subsequent isolation and analysis of the leaf oil indicated that an increased level of the assimilate was incorporated into oil of leaves exposed to the red colored light.

The elite and popular cultivars of Cymbopogon martinii were examined for genomic and expressed molecular diversity through RAPD, enzyme and SDS-PAGE protein polymorphisms. The allelic score at each locus of the enzymes as well as presence and absence profiling in RAPDs, overall occurrence of band types etc. were subjected to computation of gene diversity, expected heterozygosity, allele number per locus, and similarity matrix. These, in turn, provide inputs to derive primary account of allelic variability, genetic bases of the cultivated germplasm, putative need for gene/trait introgression from the wild or geographically diverse habitat etc. in elite selections. ‘PRC1’ possessed highest number of unique bands based on RAPD polymorphism. In variety ‘IW31245E’, diaphorase and glutamate oxaloacetate transaminase isozymes generated two unique bands as dia-III 2 and got-II 4. ‘RRL(B)77’ exhibited three unique bands; one produced by esterase as allele est-II 1 and two by malic enzyme (me-III 1,3). Only one unique band was generated by malic enzyme in variety ‘Trishna’. But sofia had three unique bands, two contributed by diaphorase (dia-II 3 and dia-II 4 and one by glutamate oxaloacetate transaminase (got-II 2). SDS-PAGE analysis revealed presence of unique polypeptide fragments (97.7 kDa to 31.6 kDa) in varieties ‘IW31245E’, ‘RRL(B)77’, ‘Tripta’, ‘Trishna’, ‘PRC1’ and var. sofia, generated as a diagnostic marker. In general, molecular distinctions associated with var. motia and var. sofia have been clearly noticed in C. martinii.

Boerhavia diffusa is extensively used in herbal medicines as well as in the Ayurvedic system, because it contains a set of clinically important compounds. In the present study, the genetic variability in Boerhavia diffusa between accessions of different geographical origin within the Indian territory is assessed through random amplified polymorphic DNA (RAPD) markers. Twenty-eight accessions of Boerhavia were screened with eighteen primers of which nine were found to be the most informative. The degree of polymorphism was found to be high in accessions collected from different places of Uttar Pradesh (Set II) in comparison to other states of India (Set I). A relatively lower level of polymorphism was recorded in accessions collected from diverse locations around Lucknow (Set III). Accessions from neighbouring geographical regions exhibited more similarity than those from distant regions (as revealed by the set I analysis). Certain diagnostic markers may be correlated with morphological character(s) such as plant type. BDL appeared most distinct and divergent from the rest of the accessions and the BDJ plant in set II also showed least similarity estimate. Fragments of 5.62 Kb and 4.47 Kb with primer GN59 was found to be unique for BDP and BD2 having ovate leaf character, whereas ovoid leaf genotype exhibited 0.79 Kb (GN34 primer) fragment. Similarly a unique band type (0.35 Kb) with primer GN83 was present in BDL and BD1 that share light pink flower. Jaccard's, and Nei and Li similarity coefficient values amongst the accessions were in the range of 0.22 to 0.89 and 0.33 to 0.93, respectively. Association of RAPD markers with the leaf characteristics, flower colour as well as with geographical locations has been made. This shows that RAPD markers are also useful for the study of genetic structure of Boerhavia populations.

Essential oil glandular trichomes are the specialized anatomical and structural characteristic of plants amassing significant quantities of commercially and pharmaceutically valuable essential oil terpenoids. Developmental dynamics of these structures together with the oil secretory process and mechanisms have a direct bearing with the secondary metabolite production, sequestration and holding potential of the producer systems. Therefore, in this study, essential-oil gland trichomes of menthol mint leaf have been stereologically analysed to discern their anatomical archetype vis-a-vis volatile oil secretion and sequestration as integrated in the overall ontogeny of leaf. Cuticular 'dehiscence' or decapping, leading to collapsing of the peltate trichomes was found to be a notable characteristic of the menthol mint oil glands. Ecophysiological, evolutionary, phytopharming and biotechnological connotations of the novel phenomenon have been hypothesized.

This review provides a summary of the physiological dynamics andregulation of essential oil production, from the literature and availableinformation on diverse volatile oil crops. Essential oil production is highlyintegrated with the physiology of the whole plant and so depends on themetabolic state and preset developmental differentiation programme of thesynthesising tissue. Essential oil productivity is ecophysiologically andenvironmentally friendly. These and other aspects of the modulation ofessentialoil production are presented, along with a brief outline of the current conceptof the relevant biosynthetic mechanisms.

A simple protocol for obtaining pure, restrictable and amplifiable megabase genomic DNA from oil free seed residue of Papaver sominiferum, an important oil seed and morphanian alkaloid yielding plant, has been developed. Fixed oil from the dry seeds was completely recovered in an organic solvent and quantified gravimetrically followed by processing of the residual biomass (defatted seed residue) for genomic DNA isolation. The isolated DNA could be cut by a range of restriction enzymes. The method enables simultaneous isolation and recovery of lipids and genomic DNA from the same test sample, thus allowing two independent analyses from a single sample.

The annual herbaceous plant, Artemisia annua L., belonging to family Asteraceae, is the: natural source of the highly potent antimalarial compound, artemisinin, besides producing valuable essential oil. The plant is at present the sole commercial source for artemisinin production since all the chemical syntheses are non-viable. Therefore, economic and practical considerations dictate that plants with maximum content of artemisinin be found and/or ways to increase their artemisinin content be sought. The key to this selection and breeding is a comprehension of chemical and genetic variability and suitable selection(s) of elites from within the available population. In the present study, RAPD analyses of selected chemotypes from a decade old introduced population in India were carried out using arbitrary primers. The RAPD data clearly indicate the distinction amongst these plants. Further, the detection of highly polymorphic profiles (97 polymorphic markers out of a total of 101 markers) suggests the existence of very high levels of genetic variation in the Indian population despite geographical isolation and opens out a strong possibility of further genetic improvement for superior artemisinin content. UPGMA analyses of RAPD and phytochemical trait data indicate that the wide phytochemical diversity is included within the genetic diversity. These results further support the prospects for selection and breeding of superior artemisinin containing lines.

Higher plants synthesize a myriad of structurally varied biologically active secondary metabolites, These compounds have been subjected to wide experimental scrutiny for various therapeutic potentials. In this review, the promise of these secondary phytochemicals as natural antimutagens has been focused upon. The compounds belonging to various chemical classes which have until now, been isolated and characterized with potential antimutagenicity are described. Besides making an extensive survey of structurally well characterized antimutagenic agents of the plant kingdom, antimutagenicity screening results of extracts of edible plants such as cereals, pulses, vegetables, oil-seeds and other health and tonic herbs reported up to date are reviewed, (C) 1998 John Wiley & Sons, Ltd.

A suitable protocol is described to obtain high molecular weight, restrictable, and amplifiable genomic DNA from the antimalarial plant Artemisia annua. The method is a CTAB procedure that includes a rapid micro-column chromatography through DE-52 ion- exchange resin. The pure consistency of the template may also offer a reliability advantage for PCR and RFLP based applications. Restriction of the purified DNA with DraI and HindIII resulted in discrete bands that may have arisen due to some repeat sequences equally spaced between the restriction site(s). Perhaps this implies a scope for direct identification of some repeat sequences such as putative species-specific probes.