Rapid solubility determination of the triterpenes oleanolic acid and ursolic acid by UV-spectroscopy in different solvents

Salvia triloba (Greek sage) has been used for the treatment of various diseases and contains two bioactive triterpene acids of major interest: oleanolic acid (OA) and ursolic acid (UA). The determination of the solubility of OA and UA in different solvents is a prerequisite to select the optimal solvent. The main goal of this work was to develop a quick method of predicting the solubility of OA/UA in different solvents to get a first indication of which solvents could be considered suitable for extraction from any plant material containing at least one of these triterpenes. A novel and simple ultra-violet spectroscopy method was developed for this purpose.The best solubilities were determined in THF, dioxane and n-butanol as well as in blends of dioxane and n-butanol.     

Relationship between Maturity Stage,Triterpenoid Content and Cytotoxicity of Sorbus intermedia (EHRH.) PERS. Fruits – A Chemometric Approach

In this study, two ursane-type triterpenes, i. e., ursolic acid (UA) and 3-O-β-acetoxyursolic acid (AUA), were isolated and quantified in Sorbus intermedia (EHRH.) PERS. fruits. UA and AUA levels differed slightly throughout fruit maturation, and both triterpenes showed similar dynamics of accumulation with the highest levels found in ripe fruits (up to 6.33±0.56 and 1.17±0.18 mg/g dw. of UA and AUA, respectively). The extracts derived from unripe fruits displayed significant cytotoxic effects against WM793, DU-145, PC3, 8505C, Caco2 and A549 cells, but no correlation between UA and AUA levels and cytotoxicity was found. On the other hand, extracts from mature fruits were not active towards almost all of the tested cell lines. The chemometric approach showed that the extracts derived from fruits harvested earlier clustered to form a clearly distanced group from extracts prepared from more-mature fruits. The extracts at higher concentrations formed separate groups, which indicated the concentration-dependent effect of these extracts on the cells.     

Molecular cloning and characterization of the global regulator LaeA in Penicillium citrinum

We have cloned and analysed a laeA gene (Pci-laeA) that may control mevastatin biosynthesis in Penicillium citrinum. The full-length Pci-laeA sequence is 1,340 bp with an ORF of 1,284 bp encoding 427 amino acids. It shows 95% identity with LaeA from P. chrysogenum. The predicted molecular mass of Pci-LaeA is 48.72 kDa with an estimated theoretical isoelectric point of 6.96. Pci-LaeA has a conserved S-adenosylmethionine binding site and a potential MlcR (a pathway specific regulator in mevastatin biosynthesis) binding site.     

影响灵芝三萜类物质液态发酵合成的关键因素研究进展

灵芝是我国名贵的食药两用型菌类,具有广泛的药用价值,其三萜类物质为灵芝中最主要的药理活性物质之一。灵芝液态发酵因具有生长周期短、环境条件可控、目标产物质量稳定及易实现规模化制备等特点而成为获得灵芝三萜类物质最有前景的方式。灵芝三萜代谢途径、发酵工艺及参数、溶解氧控制等是影响灵芝三萜类物质液态发酵合成的关键因素。本文总结了灵芝三萜生物合成的代谢途径和相关的酶(基因)、液态发酵方式和发酵参数调节的溶解氧控制这3个层面对灵芝三萜类物质生物合成的影响,并对今后的研究方向进行了展望,为液态培养灵芝三萜类物质调控及高产提供参考,也为下一步研究提供借鉴。     

Carotenoid biosynthesis genes provide evidence of geographical subdivision and extensive linkage disequilibrium in the carrot

According to the history of the cultivated carrot, root colour can be considered as a structural factor of carrot germplasm. Therefore, molecular variations of carotenoid biosynthesis genes, these being involved in colour traits, represent a good putative source of polymorphism related to diversity structure. Seven candidate genes involved in the carotenoid biosynthesis pathway have been analysed from a sample of 48 individual plants, each one from a different cultivar of carrot (Daucus carota L. ssp. sativus). The cultivars were chosen to represent a large diversity and a wide range of root colour. A high single nucleotide polymorphism (SNP) frequency of 1 SNP per 22 bp (mean π _sil = 0.020) was found on average within these genes. The analysis of genetic structure from carotenoid biosynthesis gene sequences and 17 putatively neutral microsatellites showed moderate genetic differentiation between cultivars originating from the West and the East (F _ST = 0.072), this being consistent with breeding history, but not previously evidenced by molecular tools. Surprisingly, carotenoid biosynthesis genes did not exhibit decay of LD (mean r ²  = 0.635) within the 700–1,000 bp analysed, even though a fast decay level of LD is expected in outcrossing species. The high level of intralocus LD found for carotenoid biosynthesis genes implies that candidate-gene association mapping for carrot root colour should be useful to validate gene function, but may be unable to identify precisely the causative variations involved in trait determinism. Finally this study affords the first molecular evidence of a genetic structure in cultivated carrot germplasm related to phylogeography.     

Oleanolic acid,a natural triterpenoid improves blood glucose tolerance in normal mice and ameliorates visceral obesity in mice fed a high-fat diet

Excess visceral adiposity may predispose to chronic diseases like hypertension and type 2 diabetes with a high risk for coronary artery disease. Adipose tissue secreted cytokines and oxidative stress play an important role in chronic disease progression. To combat adiposity, plant-derived triterpenes are currently receiving much attention as they possess antioxidant and anti-inflammatory properties and the ability to regulate glucose and lipid metabolism. In the search for potential antiobese compounds from natural sources, this study evaluated the effects of oleanolic acid (OA), a pentacyclic triterpene commonly present in fruits and vegetables, in glucose tolerance test and on high-fat diet (HFD)-induced obesity in mice. Adult male Swiss mice treated or not with OA (10 mg/kg) were fed a HFD during 15 weeks. Sibutramine (SIB) treated group (10 mg/kg) was included for comparison. Weekly body weights, food and water consumption were measured, and at the end of study period, the levels of blood glucose and lipids, plasma hormone levels of insulin, ghrelin and leptin, and the visceral abdominal fat content were analysed. Mice treated with OA and fed a HFD showed significantly (p < 0.05) improved glucose tolerance, decreased body weights, visceral adiposity, blood glucose, plasma lipids relative to their respective controls fed no OA. Additionally, OA treatment, while significantly elevating the plasma hormone level of leptin, decreased the level of ghrelin. However, it caused a greater decrease in plasma amylase activity than lipase. Sibutramine-treated group also manifested similar effects like OA except for blood glucose level that was not different from HFD control. These findings suggest that OA ameliorates visceral adiposity and improves glucose tolerance in mice and thus has an antiobese potential through modulation of carbohydrate and fat metabolism.     

Quantification of three triterpenic acids in dried rosemary using HPLC‐fluorescence detection and 4‐(4,5‐diphenyl‐1H‐imidazole‐2‐yl)benzoyl chloride derivatization

Functional triterpenic acids such as ursolic acid (UA), oleanolic acid (OA) and betulinic acid (BA) are representative ingredients in rosemary that may have health benefits. UA, OA and BA in rosemary extracts were derivatized with 4‐(4,5‐diphenyl‐1H‐imidazole‐2‐yl)benzoyl chloride (DIB‐Cl) and detected using HPLC‐fluorescence (FL). Dried rosemary (50 mg) was ground, added to 3 ml of ethanol, sonicated for 40 min, then the sample solution was added to a mixture of 1% trimethylamine and 1 mM DIB‐Cl in acetonitrile. The mixture was settled for 5 min at room temperature, then the DIB‐triterpenic acid derivatives were separated using a Wakopak Handy ODS column (250 × 4.6 mm, 6 μm) eluted with 25 mM acetate buffer (pH 4.5)/methanol/acetonitrile (= 8:10:82 v/v/v%). The fluorescence intensity of the eluent was monitored at 365 (λ_ex) and 490 nm (λ_em) and the maximum retention time of the derivatives was 30 min. Calibration curves constructed using rosemary extract spiked with standards showed good linearity (r ≥ 0.997) in the range 2.5–100 ng/ml. The detection limits at 3σ for internal BA, UA and OA peaks in rosemary extract were 0.2, 0.4 and 0.5 ng/ml, respectively. This method was used to quantify BA, UA and OA in commercially available dried rosemary products.     

Analysis of pYC2, a cryptic plasmid in <Emphasis Type="Italic">Lactobacillus sakei</Emphasis> BM5 isolated from kimchi

Analysis of the structural properties of pYC2, a cryptic plasmid from Lactobacillus sakei BM5 isolated from kimchi, determined its length as 1,970 bp with a G + C content of 34%. The double-strand origin (dso) and single-strand origin (sso) of rolling-circle replicating plasmids were found in the nucleotide sequence of the pYC2 plasmid. Sequence analysis of pYC2 revealed that ORF 1 and ORF 2 showed high homology with the Cop and Rep proteins encoded by the pMV158 family of plasmids replicating via the rolling-circle mechanism. pYC2 also replicates by this mechanism, as confirmed by Southern hybridization analysis.     

Engineering triterpene metabolism in the oilseed of Arabidopsis thaliana

Squalene and botryococcene are linear, hydrocarbon triterpenes that have industrial and medicinal values. While natural sources for these compounds exist, there is a pressing need for robust, renewable production platforms. Oilseeds are an excellent target for heterologous production because of their roles as natural storage repositories and their capacity to produce precursors from photosynthetically‐derived carbon. We generated transgenic Arabidopsis thaliana plants using a variety of engineering strategies (subcellular targeting and gene stacking) to assess the potential for oilseeds to produce these two compounds. Constructs used seed‐specific promoters and evaluated expression of a triterpene synthase alone and in conjunction with a farnesyl diphosphate synthase (FPS) plus 1‐deoxyxylulose 5‐phosphate synthase (DXS). Constructs directing biosynthesis to the cytosol to harness isoprenoid precursors from the mevalonic acid (MVA) pathway were compared to those directing biosynthesis to the plastid compartment diverting precursors from the methylerythritol phosphate (MEP) pathway. On average, the highest accumulation for both compounds was achieved by targeting the triterpene synthase, FPS and DXS to the plastid (526.84 μg/g seed for botryococcene and 227.30 μg/g seed for squalene). Interestingly, a higher level accumulation of botryococcene (a non‐native compound) was observed when the biosynthetic enzymes were targeted to the cytosol (>1000 μg/g seed in one line), but not squalene (natively produced in the cytosol). Not only do these results indicate the potential of engineering triterpene accumulation in oilseeds, but they also uncover some the unique regulatory mechanisms controlling triterpene metabolism in different cellular compartments of seeds.     

Molecular cloning,characterization and function analysis of the gene encoding HMG-CoA reductase from <Emphasis Type="Italic">Euphorbia Pekinensis</Emphasis> Rupr

A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.     

Engineering linear,branched‐chain triterpene metabolism in monocots

Triterpenes are thirty‐carbon compounds derived from the universal five‐carbon prenyl precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Normally, triterpenes are synthesized via the mevalonate (MVA) pathway operating in the cytoplasm of eukaryotes where DMAPP is condensed with two IPPs to yield farnesyl diphosphate (FPP), catalyzed by FPP synthase (FPS). Squalene synthase (SQS) condenses two molecules of FPP to generate the symmetrical product squalene, the first committed precursor to sterols and most other triterpenes. In the green algae Botryococcus braunii, two FPP molecules can also be condensed in an asymmetric manner yielding the more highly branched triterpene, botryococcene. Botryococcene is an attractive molecule because of its potential as a biofuel and petrochemical feedstock. Because B. braunii, the only native host for botryococcene biosynthesis, is difficult to grow, there have been efforts to move botryococcene biosynthesis into organisms more amenable to large‐scale production. Here, we report the genetic engineering of the model monocot, Brachypodium distachyon, for botryococcene biosynthesis and accumulation. A subcellular targeting strategy was used, directing the enzymes (botryococcene synthase [BS] and FPS) to either the cytosol or the plastid. High titres of botryococcene (>1 mg/g FW in T₀ mature plants) were obtained using the cytosolic‐targeting strategy. Plastid‐targeted BS + FPS lines accumulated botryococcene (albeit in lesser amounts than the cytosolic BS + FPS lines), but they showed a detrimental phenotype dependent on plastid‐targeted FPS, and could not proliferate and survive to set seed under phototrophic conditions. These results highlight intriguing differences in isoprenoid metabolism between dicots and monocots.     

Underutilized <Emphasis Type="Italic">Annona</Emphasis> Species from the Brazilian Cerrado and Amazon Rainforest: A Study on Fatty Acids Profile and Yield of Seed Oils

Annona (Annonaceae) is an important source of fruits in the Brazilian Cerrado and the Amazon Rainforest. Some Annona species are widely commercialized as fresh fruit or as frozen pulp. Seeds are accustomedly discarded. Our main goal was to analyze fatty acids profile from seeds of A. crassiflora and A. coriacea from Cerrado, A. montana from Amazon Forest, and three cultivars (A. cherimola cv. Madeira, A. cherimola x A. squamosa cv. Pink’s Mammonth and A. cherimola x A. squamosa cv. Gefner). The total oil yield ranged between 20 and 42% by weight of dry mass. The A cherimola x A. squamosa cv. Gefner has significantly higher total lipid yield than all other samples. 100 g of fruit of this species present 6-8 g of seeds. Considering the fruit production of Chile (over 221 ton of fruits/year), more than 1300 ton of seed/year could be obtained, which could provide at least 200 ton of seed oil. Oleic acid was predominant for most samples, but for A. montana linoleic acid was the most abundant FA. Phenotypic variation on FAME profile was observed. These new data are an urgent requirement for supporting conservation programs, mainly for Cerrado areas in Brazil.     

Ascorbate content of wheat leaves is not determined by maximal l-galactono-1,4-lactone dehydrogenase (GalLDH) activity under drought stress

Although ascorbic acid (AA) is a high-abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l -galactono-1,4-lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well-watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.