Characterization of a CrPME indicates its possible role in determining vindoline accumulation in Catharanthus roseus leaves.

The leaf-specific Catharanthus roseus alkaloid, vindoline, is the major bottleneck precursor in the production of scarce and costly anticancer bisindoles (vincristine and vinblastine). The final steps of its biosynthesis and storage occur in the laticifers. Earlier, we have shown that vindoline content is directly related to laticifer number. Pectin remodeling enzymes, like pectin methylesterase (PME), are known to be involved in laticifer development. A search in the croFGD yielded a leaf-abundant CrPME isoform that co-expressed with a few vindoline biosynthetic genes. Full-length cloning, tissue-specific expression profiling, and in silico analysis of CrPME were carried out. It was found to possess all the specific characteristics of a typical plant PME. Transient silencing (through VIGS) and overexpression of CrPME in C. roseus indicated a direct relationship between its expression and vindoline content. Comparative analysis of transcript abundance and enzyme activity in three familial C. roseus genotypes differing significantly in their vindoline content and laticifer count (CIM-Sushil > Dhawal > Nirmal) also corroborated the positive relationship of CrPME expression with vindoline content. This study highlights the possible role of CrPME, a cell wall remodeling enzyme, in modulating laticifer-associated secondary metabolism.

Chemico-nutritional characterization and anti-inflammatory potential of Chirabilva (Holoptelea integrifolia Roxb.) seed: alternate source of protein supplement and fatty acids.

Exploring unconventional protein sources can be an alternative strategy to meet the deficiency. The seeds of Chirabilva (Holoptelea integrifolia Roxb., Family- Ulmaceae) are eaten raw by the ethnic communities of Southeast Asian countries. The present study assessed the chemical, nutritional, and biological potential of the seeds (HIS) and pericarp (HISP) of H. integrifolia. The seeds contain mainly fixed and very few essential oils. The fixed oil of HIS is composed primarily of unsaturated oleic (47%) and saturated palmitic (37%) acids. The HIS are exceptional due to a high content of lipid (50%), protein (24%), carbohydrates (19%), fiber (4%), and anti-nutritional components within permissible limits. The high content (in mg/Kg) of phosphorus (6000), magnesium (422), Calcium (279), and essential nutrients (Ni, Co, Zn, Fe, Cu, Mn, and Cr) in the range of (0.04-6.69) were observed. The moderate anti-oxidant potential of HISP was evident in single electron transfer in-vitro assays. Moreover, HISP extract and HIS solvent-extracted fixed oil showed anti-inflammatory action in lipopolysaccharide-induced HaCaT cells by significantly attenuating pro-inflammatory cytokines (TNF-α) without causing cytotoxicity. Results support de-oiled HIS cake as an alternative source of a high-protein diet and its oil with anti-inflammatory attributes for topical applications.

Temperature-induced lipocalin-mediated membrane integrity: Possible implications for vindoline accumulation in Catharanthus roseus leaves.

Plant lipocalins perform diverse functions. Recently, allene oxide cyclase, a lipocalin family member, has been shown to co-express with vindoline pathway genes in Catharanthus roseus under various biotic/abiotic stresses. This brought focus to another family member, a temperature-induced lipocalin (CrTIL), which was selected for full-length cloning, tissue-specific expression profiling, in silico characterization, and upstream genomic region analysis for cis-regulatory elements. Stress-mediated variations in CrTIL expression were reflected as disturbances in cell membrane integrity, assayed through measurement of electrolyte leakage and lipid peroxidation product, MDA, which implicated the role of CrTIL in maintaining cell membrane integrity. For ascertaining the function of CrTIL in maintaining membrane stability and elucidating the relationship between CrTIL expression and vindoline content, if any, a direct approach was adopted, whereby CrTIL was transiently silenced and overexpressed in C. roseus. CrTIL silencing and overexpression confirmed its role in the maintenance of membrane integrity and indicated an inverse relationship of its expression with vindoline content. GFP fusion-based subcellular localization indicated membrane localization of CrTIL, which was in agreement with its role in maintaining membrane integrity. Altogether, the role of CrTIL in maintaining membrane structure has possible implications for the intracellular sequestration, storage, and viability of vindoline.

Dual targeted 2-Benzylideneindanone pendant hydroxamic acid group exhibits selective HDAC6 inhibition along with tubulin stabilization effect.

Abnormal epigenetics has been recognised as an early event in tumour progression and aberrant acetylation of lysine in particular has been understood in tumorigenesis. Therefore, it has become an attractive target for anticancer drug development. However, HDAC inhibitors have limited success due to toxicity and drug resistance concerns. Present study deals with design and synthesis of bivalent indanone based HDAC6 and antitubulin ligands as anticancer agents. Two of the analogues 9 and 21 exhibited potent antiproliferative activities (IC50, 0.36-3.27 µM) and high potency against HDAC 6 enzyme. Compound 21 showed high selectivity against HDAC 6 while 9 exhibited low selectivity. Both the compounds also showed microtubule stabilization effects and moderate anti-inflammatory effect. Dual targeted anticancer agents with concomitant anti-inflammatory effects will be more attractive clinical candidates in future.

Liquiritigenin, isoliquiritigenin rich extract of glycyrrhiza glabra roots attenuates inflammation in macrophages and collagen-induced arthritis in rats.

Liquiritigenin (LTG) and its bioprecursor isoliquiritigenin(ISL), the main bioactives from roots of Glycyrrhiza genus are progressively documented as a potential pharmacological agent for the management of chronic diseases. The aim of this study was to evaluate the pharmacological potential of liquiritigenin, isoliquiritigenin rich extract of Glycyrrhiza glabra roots (IVT-21) against the production of pro-inflammatory cytokines from activated macrophages as well as further validated the efficacy in collagen-induced arthritis model in rats. We also performed the safety profile of IVT-21 using standard in-vitro and in-vivo assays. Results of this study revealed that the treatment of IVT-21 and its major bioactives (LTG, ISL) was able to reduce the production of pro-inflammatory cytokines (TNF-α, IL-6) in LPS-activated primary peritoneal macrophages in a dose-dependent manner compared with vehicle-alone treated cells without any cytotoxic effect on macrophages. In-vivo efficacy profile against collagen-induced arthritis in Rats revealed that oral administration of IVT-21 significantly reduced the arthritis index, arthritis score, inflammatory mediators level in serum. IVT-21 oral treatment is also able to reduce the NFкB-p65 expression as evidence of immunohistochemistry in knee joint tissue and mRNA level of pro-inflammatory cytokines in paw tissue in a dose-dependent manner when compared with vehicle treated rats. Acute oral toxicity profile of IVT-21 demonstrated that it is safe up to 2000 mg/kg body weight in experimental mice. This result suggests the suitability of IVT-21 for further study in the management of arthritis and related complications.

Overexpression of chrysanthemyl diphosphate synthase (CDS) gene in Tagetes erecta leads to the overproduction of pyrethrin.

Pyrethrins are widely accepted as natural insecticides and offers several advantages of synthetic compounds, i.e., rapidity of action, bioactivity against a wide range of insects, comparatively lesser costs and the like. A significant source of pyrethrin is Chrysanthemum cinerariaefolium; cultivated in restricted areas, as a result; natural pyrethrins are not produced in a large amount that would meet the ongoing global market demand. However, increasing its content and harnessing the desired molecule did not attract much attention. To enhance the production of pyrethrins in Tagetes erecta, the Chrysanthemyl diphosphate synthase (CDS) gene was overexpressed under the promoter CaMV35S. Hypocotyls were used as explant for transformation, and direct regeneration was achieved on MS medium with 1.5 mg L-1 BAP and 5.0 mg L-1 GA3. Putative transgenics were screened on 10 mgL-1 hygromycin. After successful regeneration, screening and rooting process, the transgenic plants were raised inside the glass house and PCR amplification of CDS and HYG-II was used to confirm the transformation. Biochemical analysis using HPLC demonstrated the expression levels of the pyrethrin, which was approx. twenty-six fold higher than the non-transformed Tagetes plant.

Quantification of genetic variability and stable genotype selection over the years in the germplasm of critically endangered Prishanparni (Uraria picta Desv.).

Prishanparni (Uraria picta Desv.), a critically endangered annual shrub belonging to the family 'Papillionaceae'. It is widely distributed throughout India, Sri Lanka, Bangladesh, Tropical Africa, Malay Islands, and the Philippines. The consistent performances of U. picta accessions based on ten economic traits studied were identified as P-12, P-16, P-21, P-22, P-31, P-47, and P-48. These accessions could be used for commercial cultivation in northern Indian plains. Among the total twenty-three studied accessions P-50, P-21, P-48, and P-47 were found superior for rhoifolin content in their aerial as well as root part, which may have various therapeutic potentials used in traditional and modern systems of medicines. These accessions can be exploited for commercial cultivation or in a hybridization program for further crop improvement. Wide range cultivation of the selected accessions in the Indo-Gangetic plains will fit in the existing cropping systems of this region, resulting in comparatively better supplementation of herb to the pharmaceutical and herbal drug industries and reducing the pressure on the wild populations.

A plastid-localized bona fide geranylgeranyl diphosphate synthase plays a necessary role in monoterpene indole alkaloid biosynthesis in Catharanthus roseus.

In plants, geranylgeranyl diphosphate (GGPP, C20 ) synthesized by GGPP synthase (GGPPS) serves as precursor for vital metabolic branches including specialized metabolites. Here, we report the characterization of a GGPPS (CrGGPPS2) from the Madagascar periwinkle (Catharanthus roseus) and demonstrate its role in monoterpene (C10 )-indole alkaloids (MIA) biosynthesis. The expression of CrGGPPS2 was not induced in response to methyl jasmonate (MeJA), and was similar to the gene encoding type-I protein geranylgeranyltransferase_ß subunit (CrPGGT-I_ß), which modulates MIA formation in C. roseus cell cultures. Recombinant CrGGPPS2 exhibited a bona fide GGPPS activity by catalyzing the formation of GGPP as the sole product. Co-localization of fluorescent protein fusions clearly showed CrGGPPS2 was targeted to plastids. Downregulation of CrGGPPS2 by virus-induced gene silencing (VIGS) significantly decreased the expression of transcription factors and pathway genes related to MIA biosynthesis, resulting in reduced MIA. Chemical complementation of CrGGPPS2-vigs leaves with geranylgeraniol (GGol, alcoholic form of GGPP) restored the negative effects of CrGGPPS2 silencing on MIA biosynthesis. In contrast to VIGS, transient and stable overexpression of CrGGPPS2 enhanced the MIA biosynthesis. Interestingly, VIGS and transgenic-overexpression of CrGGPPS2 had no effect on the main GGPP-derived metabolites, cholorophylls and carotenoids in C. roseus leaves. Moreover, silencing of CrPGGT-I_ß, similar to CrGGPPS2-vigs, negatively affected the genes related to MIA biosynthesis resulting in reduced MIA. Overall, this study demonstrated that plastidial CrGGPPS2 plays an indirect but necessary role in MIA biosynthesis. We propose that CrGGPPS2 might be involved in providing GGPP for modifying proteins of the signaling pathway involved in MIA biosynthesis.

Molecular insights into enhanced resistance of Papaver somniferum against downy mildew by application of endophyte bacteria Microbacterium sp. SMR1.

Downy mildew is one of the most serious diseases of Papaver somniferum. Endophytes isolated from different parts of P. somniferum were screened for their ability to enhance resistance against downy mildew caused by the obligate biotrophic oomycete Peronospora meconopsidis. Two endophytes (SMR1 and SMR2) reduced the downy mildew on three P. somniferum genotypes (Sampada, J-16, and I-14). SMR1 (Microbacterium sp.) also enhanced the resistance of P. somniferum against downy mildew under field conditions. The biochemical markers of plant susceptibility under biotic stresses (proline and malondialdehyde) were found to be reduced in P. somniferum upon SMR1 treatment. To understand the mechanisms underlying the enhanced resistance to downy mildew in SMR1 endophyte-treated P. somniferum genotype J-16, we compared the expression profiles using the next-generation RNA sequencing approach between P. somniferum pretreated with SMR1 and untreated endophyte-free control plants following exposure to downy mildew pathogen. Comparative transcriptome analysis revealed differential expression of transcripts belonging to broad classes of signal transduction, protein modification, disease/defense proteins, transcription factors, and phytohormones in SMR1-primed P. somniferum after infection with downy mildew pathogen. Furthermore, enhanced salicylic acid content was observed in SMR1-primed P. somniferum after exposure to downy mildew pathogen. This study sheds light on molecular mechanisms underlying enhanced resistance to downy mildew in SMR1-primed P. somniferum. Finally, we propose that the SA-dependent defense pathway, the hallmark of systemic acquired resistance, is activated in SMR1-primed P. somniferum, triggering the endophyte-induced resistance.

Biochar amendment reduced the risk associated with metal uptake and improved metabolite content in medicinal herbs.

Contaminations of heavy metals such as lead (Pb) and cadmium (Cd) in medicinal plants (MPs) not only restrict their safe consumption due to health hazards but also lower their productivity. Biochar amendments in the soil are supposed to immobilize the toxic metals, improve the soil quality and agricultural productivity. However, the impact of biochar on growth attributes, metal accumulation, pharmacologically active compounds of MPs, and health risk is less explored. An experiment was performed on three medicinal plants (Bacopa monnieri (L.), Andrographis paniculata (Burmf.) Nees, and Withaniasomnifera (L.)) grown in a greenhouse in soil co-contaminated with Pb and Cd (at two concentrations) without and with biochar amendments (2 and 4% application rates). The fractionation of Pb and Cd, plant growth parameters, stress enzymes, photosynthetic capacity, pharmacologically active compounds, nutrient content, uptake and translocation of metals, antioxidant activities, and metabolite content were examined in the three MPs. The accumulation of Pb and Cd varied from 3.25-228 mg kg1 and 1.29-20.2 mg kg-1 , respectively, in the three MPs, while it was reduced to 0.08-18 mg kg-1 and 0.03-6.05 mg kg-1 upon biochar treatments. Plants grown in Pb and Cd co-contaminated soil had reduced plant biomass (5-50% depending on the species) compared to control and a deleterious effect on photosynthetic attributes and protein content. However, biochar amendments significantly improved plant biomass (21-175%), as well as photosynthesis attributes, chlorophyll, and protein contents. Biochar amendments in Pb and Cd co-contaminated soil significantly reduced the health hazard quotient (HQ) estimated for the consumption of these medicinal herbs grown on metal-rich soil. An enhancement in secondary metabolite content and antioxidant properties was also observed upon biochar treatments. These multiple beneficial effects of biochar supplementation in Pb and Cd co-contaminated soil suggested that a biochar amendment is a sustainable approach for the safe cultivation of MPs. This article is protected by copyright. All rights reserved.

Design, synthesis and broad spectrum antibreast cancer activity of diarylindoles via induction of apoptosis in aggressive breast cancer cells.

Breast cancer is the second leading cause of cancer deaths in women with significant morbidity and mortality. Present study describes design, synthesis and detailed pharmacology of indole derivatives exhibiting remarkable broad spectrum antiproliferative activity against breast cancer cells. Detailed mechanistic evaluations confirmed induction of G0/G1 arrest, apoptosis induction, loss of mitochondrial integrity, enhanced ROS generation, autophagy, estrogen receptor ß-transactivation and increased tubulin polymerization. In in-vivo efficacy studies in rodent model, these indole derivatives induced significant regression in mice mammary tumour on 21 days daily oral dose. Moreover, compounds 19 and 23 were safe in Swiss albino mice in safety studies. These diarylindoles may further be optimized for better efficacy.

Hesperidin-rich ethanol extract from waste peels of Citrus limetta mitigates rheumatoid arthritis and related complications.

The aim of this study is to explore the possible pharmacological effects of fruit waste that may have a key role in converting the fruit waste into pharmaceutical agents. Citrus limetta (Rutaceae) is an important commercial citrus fruit crops used by juice processing industries. C. limetta peels are perishable waste material, which creates a big challenge in juice processing industries. Initial pharmaco-chemical profile of peels' extracts revealed that the ethanol extract (ClPs) has promising anti-inflammatory activity and rich in hesperidin content. In vivo experimental pharmacology profile of ClPs against arthritis and related complications revealed that oral administration of ClPs significantly reduced the arthritis score and arthritis index in elbow and knee joints against collagen-induced arthritis (CIA) in rats. Biochemical parameters include pro-inflammatory cytokines (TNF-α, IL-6, and IL-17A), and C-RP level in blood serum of CIA rats further confirmed the anti-arthritic profile of ClPs. Further individual experiments related to arthritis-related complications in experimental animals demonstrated the analgesic, anti-inflammatory, and antipyretic potential of ClPs in dose-dependent manner. The result of this study suggests the suitability of ClPs as a drug-like candidate for further investigation toward the management of arthritis and related complications.

Cliv-92-Loaded Glycyrrhetinic Acid-Modified Chitosan Nanoparticles for Enhanced Hepatoprotection-Preparation, Characterization, and In Vivo Evaluation.

Cliv-92 is a mixture of three structurally similar coumarinolignoids and a proven hepatoprotective agent. Low aqueous solubility and poor bioavailability are notable hindrances for its further use. Therefore, glycyrrhetinic acid-linked chitosan nanoparticles loaded with Cliv-92 were prepared for active targeting to the liver. The nanoparticles were prepared by the ionic gelation method to avoid the use of toxic solvents/rigorous agitation. The method of preparation was optimized using a central composite design with independent variables, namely polymer: drug ratio (3:1, w/w), crosslinker concentration (0.5%), and stirring speed (750 rpm). The optimized nanoparticles had a mean particle size of 185.17 nm, a polydispersity index of 0.41, a zeta potential of 30.93 mV, and a drug loading of 16.30%. The prepared formulation showed sustained release of approximately 63% of loaded Cliv-92 over 72 h. The nanoparticles were freeze-dried for long-term storage and further characterized. The formulation was found to be biocompatible for parenteral delivery. In vivo imaging study showed that optimized nanoparticles were preferentially accumulated in the liver and successfully targeting the liver. The present study successfully demonstrated the improved pharmacokinetic properties (≈12% relative bioavailability) and efficacy profile (evidenced by in vivo and histopathological studies) of fabricated Cliv-92 nanoparticles.

Biochemical characterization and spatio-temporal analysis of the putative L-DOPA pathway in Mucuna pruriens.

MAIN CONCLUSION: Transcriptome analysis and biochemical characterization of the putative l-3,4-dihydroxyphenylalanine (l-DOPA) pathway in Mucuna pruriens (L.) DC have been performed. Spatio-temporal quantification of the putative l-DOPA biosynthetic pathway genes and its correlation with respective metabolites was established. l-tyrosine, l-DOPA, and dopamine from all plant parts were quantified. The de novo transcriptome analysis was performed using leaves of the selected M. pruriens mutant T-IV-9 during maturity. The putative L-DOPA pathway and its regulatory genes were retrieved from transcriptome data and the L-DOPA pathway was biochemically characterized. The spatial and temporal gene expression for the L-DOPA pathway was identified with respect to the chemical constituents. L-tyrosine, L-DOPA, and dopamine contents were highest in leaves during maturity (about 170-day-old plants). The polyphenol oxidase (PPO) was highly expressed in tender stems (230-fold higher as compared to seeds) as well as a high L-DOPA content. The PPO gene was highly expressed in leaves (3367.93 in FPKM) with a 79-fold increase compared to control plants during maturity. L-DOPA was found in every part with varied levels. The highest L-DOPA content was found in mature dried seed (3.18-5.8%), whereas the lowest amount was recorded in mature and dried leaves. The reproductive parts of the plant had a higher amount of L-DOPA content (0.9-5.8%) compared to the vegetative parts (0.2-0.91%). Various amino acid transporters and permeases were expressed in M. pruriens. The transcripts of dopa decarboxylase (DDC) were found in almost all parts of the plant, but its higher content was limited to the leaf.

A novel synthesis of 2-arylbenzimidazoles in molecular sieves-MeOH system and their antitubercular activity.

Arylbenzimidazoles have been synthesized as antimycobacterial agents. An efficient synthesis has been developed for 2-arylbenzimidazoles from o-phenylenediamines and aromatic aldehydes in molecular sieves-methanol system. The methodology is straightforward to get 2-arylbenzimidazoles (3a-3z) in excellent yields with high chemoselectivity over 2-aryl-1-benzylbenzimidazoles (4a-4z). All these benzimidazole analogues were evaluated against M. tuberculosis in BACTEC radiometric assay. The compounds 4y and 4z exhibited potential antitubercular activity against M. tuberculosis H37RV, MIC at 16 µM and 24 µM respectively. The best compound of the series i.e. compound 4y was well tolerated by Swiss-albino mice in acute oral toxicity. Compound 4y possessing a diarylbenzimidazole core, can further be optimized for better activity.

Cloning and functional characterization of quinolinic acid phosphoribosyl transferase (QPT) gene of Nicotiana tabacum.

The quinolinate phosphoribosyl transferase (QPT) is a key enzyme that converts quinolinic acid into nicotinic acid mononucleotide. The QPT gene plays an essential role in the pyridine nucleotide cycle as well as in the biosynthetic pathway of the alkaloid nicotine. However, a clear role for QPT is yet to be characterized to validate the actual function of this gene in planta. In this study, an RNA interference (RNAi) approach was used to reveal the functional role of QPT. Transformation and analysis of the hairy roots (HRs) of the Nicotiana leaf explants was used, followed by plant regeneration and analysis. High-performance liquid chromatography (HPLC) analysis of the HRs and of the regenerated plants both revealed altered alkaloid biosynthetic cycle, with a substantially reduced content of nicotine and anabasine. The transgenic plants exhibited a significantly altered phenotype and growth pattern. Also, silencing of QPT led to a decrease in chlorophyll content, maximum quantum efficiency of PSII, net CO2 assimilation and starch content. Results clearly demonstrated that QPT was not only involved in the biosynthetic pathway of the alkaloids but also affected plant growth and development. Our results provide information to be considered when trying to engineer the secondary metabolite quality and quantity.

Endophytes of opium poppy differentially modulate host plant productivity and genes for the biosynthetic pathway of benzylisoquinoline alkaloids.

MAIN CONCLUSION: Endophytes reside in different parts of the poppy plant and perform the tissue-specific functions. Most leaf endophytes modulate photosynthetic efficiency, plant growth, and productivity while capsule endophytes modulate alkaloid biosynthesis. Endophytes promote plant growth, provide protection from environmental stresses and are the source of important secondary metabolites. Here, we established that the endophytes of opium poppy Papaver somniferum L. may play a role in the modulation of plant productivity and benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 22 endophytes isolated from leaves, roots, capsules and seeds of the poppy plants were identified. Isolated endophytes were used to inoculate the endophytes free poppy seeds and screened for their ability to improve plant productivity and BIA production. It was evident that the endophytes from leaf were involved in improving photosynthetic efficiency, and thus crop growth and yield and the endophytes from capsule were involved in enhancing BIA biosynthesis. Capsule endophytes of alkaloid-rich P. somniferum cv. Sampada enhanced BIA production even in alkaloid-less cv. Sujata. Expression study of the genes involved in BIA biosynthesis conferred the differential regulation of their expression in the presence of capsule endophytes. The capsule endophyte SM1B (Acinetobacter) upregulated the expression of the key genes for the BIA biosynthesis except thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). On the other hand, another capsule endophyte SM3B (Marmoricola sp.) could upregulate both T6ODM and CODM. Colonization of poppy plant by endophytes isolated from leaves, roots and capsules found to be higher in their respective plant parts confirmed their tissue-specific role. Overall, the results demonstrate the specific role of endophytes in the modulation of host plant productivity and BIA production.

Methyl jasmonate-elicited transcriptional responses and pentacyclic triterpene biosynthesis in sweet basil.

Sweet basil (Ocimum basilicum) is well known for its diverse pharmacological properties and has been widely used in traditional medicine for the treatment of various ailments. Although a variety of secondary metabolites with potent biological activities are identified, our understanding of the biosynthetic pathways that produce them has remained largely incomplete. We studied transcriptional changes in sweet basil after methyl jasmonate (MeJA) treatment, which is considered an elicitor of secondary metabolites, and identified 388 candidate MeJA-responsive unique transcripts. Transcript analysis suggests that in addition to controlling its own biosynthesis and stress responses, MeJA up-regulates transcripts of the various secondary metabolic pathways, including terpenoids and phenylpropanoids/flavonoids. Furthermore, combined transcript and metabolite analysis revealed MeJA-induced biosynthesis of the medicinally important ursane-type and oleanane-type pentacyclic triterpenes. Two MeJA-responsive oxidosqualene cyclases (ObAS1 and ObAS2) that encode for 761- and 765-amino acid proteins, respectively, were identified and characterized. Functional expressions of ObAS1 and ObAS2 in Saccharomyces cerevisiae led to the production of ß-amyrin and α-amyrin, the direct precursors of oleanane-type and ursane-type pentacyclic triterpenes, respectively. ObAS1 was identified as a ß-amyrin synthase, whereas ObAS2 was a mixed amyrin synthase that produced both α-amyrin and ß-amyrin but had a product preference for α-amyrin. Moreover, transcript and metabolite analysis shed light on the spatiotemporal regulation of pentacyclic triterpene biosynthesis in sweet basil. Taken together, these results will be helpful in elucidating the secondary metabolic pathways of sweet basil and developing metabolic engineering strategies for enhanced production of pentacyclic triterpenes.

Chemical composition and acetylcholinesterase inhibitory activity of Artemisia maderaspatana essential oil.

CONTEXT: To date, there are no reports to validate the Indian traditional and folklore claims of Artemisia maderaspatana L. (syn. Grangea maderaspatana L.) (Asteraceae) for the treatment of Alzheimer's disease. OBJECTIVE: The present study characterizes the volatile components (non-polar compounds) of A. maderaspatana and evaluates its acetylcholinesterase inhibition potential. MATERIALS AND METHODS: The essential oils (yield 0.06% v/w) were obtained from fresh aerial part of A. maderaspatana. The characterization of volatile components (non-polar compounds) was performed by GC-MS data and with those of reference compounds compiled in the spectral library of in-house database. The in vitro acetylcholinesterase (AChE) inhibition of the volatile organic constituents (VOC's) of A. maderaspatana aerial part was evaluated in varying concentration ranges (0.70-44.75 µg/mL) with Ellman's method. RESULTS: The major components were α-humulene (46.3%), ß-caryophyllene (9.3%), α-copaene (8.2%), ß-myrcene (4.3%), Z(E)-α-farnesene (3.7%), and calarene (3.5%). Chemical variability among other Artemisia spp. from different climatic regions of India and countries namely Iran and France was observed. The experimental results showed that diverse volatile organic constituents of A. maderaspatana have significant acetylcholinesterase inhibitory activity (an IC50 value of 31.33 ± 1.03 µg/mL). This is the first report on the inhibition of acetylcholinesterase properties of essential oil of A. maderaspatana obtained from fresh aerial part. CONCLUSIONS: The present results indicate that essential oil of A. maderaspatana isolated from the northern region of India could inhibit AChE moderately. Therefore, the possibility of novel AChE inhibitors might exist in VOCs of this plant.

Sterol partitioning by HMGR and DXR for routing intermediates toward withanolide biosynthesis.

Withanolides biosynthesis in the plant Withania somnifera (L.) Dunal is hypothesized to be diverged from sterol pathway at the level of 24-methylene cholesterol. The conversion and translocation of intermediates for sterols and withanolides are yet to be characterized in this plant. To understand the influence of mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways on sterols and withanolides biosynthesis in planta, we overexpressed the WsHMGR2 and WsDXR2 in tobacco, analyzed the effect of transient suppression through RNAi, inhibited MVA and MEP pathways and fed the leaf tissue with different sterols. Overexpression of WsHMGR2 increased cycloartenol, sitosterol, stigmasterol and campesterol compared to WsDXR2 transgene lines. Increase in cholesterol was, however, marginally higher in WsDXR2 transgenic lines. This was further validated through transient suppression analysis, and pathway inhibition where cholesterol reduction was found higher due to WsDXR2 suppression and all other sterols were affected predominantly by WsHMGR2 suppression in leaf. The transcript abundance and enzyme analysis data also correlate with sterol accumulation. Cholesterol feeding did not increase the withanolide content compared to cycloartenol, sitosterol, stigmasterol and campesterol. Hence, a preferential translocation of carbon from MVA and MEP pathways was found differentiating the sterols types. Overall results suggested that MVA pathway was predominant in contributing intermediates for withanolides synthesis mainly through the campesterol/stigmasterol route in planta.