A new anti-austerity agent, 4'-O-methylgrynullarin from Derris scandens induces PANC-1 human pancreatic cancer cell death under nutrition starvation via inhibition of Akt/mTOR pathway.

An ethanolic extract of Derris scandens flowers showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived condition, with a PC50 value of 0.7 µg/mL. Phytochemical investigation of this active extract led to the isolation of four prenylated isoflavones (1-4) including a new compound named 4'-O-methylgrynullarin (1). The structure elucidation of the new compound was achieved by HRFABMS and NMR spectroscopic analysis. The isolated compounds exhibited potent anti-austerity activity against four different human pancreatic cancer cell lines under nutrient-deprived conditions. The new compound 4'-O-methylgrynullarin (1) was also found to inhibit PANC-1 cell migration and colony formation under nutrient-rich condition. Mechanistically, compound 1 inhibited key survival proteins in the Akt/mTOR signaling pathway. Therefore, 4'-O-methylgrynullarin (1) can be considered as a potential lead compound for the anticancer drug development based on the anti-austerity strategy.

Nine prenylated acylphloroglucinols with potential anti-depressive and hepatoprotective activities from Hypericum scabrum.

In our screening program for new biologically active secondary metabolites, nine new polycyclic polyprenyled acylphloroglucinols, hyperscabins D-L, together with three known compounds, were obtained from the aerial parts of Hypericum scabrum. The chemical structures of 1-9 were characterized by extensive spectroscopic analyses, nuclear magnetic resonance calculation with DP4+ probability analysis, and the electronic circular dichroism spectra were calculated. Compound 1 was an unusual prenylated acylphloroglucinol decorated with a 5-oxaspiro [4,5] deca-1,9-dione skeleton. Compound 2 was a newly identified spirocyclic polyprenylated acylphloroglucinol possessing a rare 5,5-spiroketal segment. Compounds 3, 8, and 10 (10 µM) exhibited pronounced hepatoprotective activity against d-galactosamine-induced WB-F344 cell damage in vitro assays. All test compounds (1, 3, and 7-12) demonstrated potential inhibitory effects at 10 µM against noradrenalinet ([3H]-NE) reuptake in rat brain synaptosome.

Chemical Profile and Use of the Peat as an Adsorbent for Extraction of Volatile Compounds from Leaves of Geranium (Pelargonium graveolens L' Herit).

Volatile organic compounds (VOCs) from leaves of geranium (Pelargonium graveolens L' Herit) were extracted by dynamic headspace using Porapak Q (HSD-P) as adsorbent and peat, a novel adsorbent in the extraction of plant volatiles, analyzed by gas chromatography-mass spectrometry (GC/MS) and gas chromatography-flame ionization (GC/FID), and the results were compared with those obtained by hydrodistillation (HD). The yield volatiles changed with the extraction method. HD was more efficient for extracting linalool (11.19%) and citronellyl formate (9.41%). Citronellol (28.06%), geraniol (38.26%) and 6,9-guaiadiene (9.55%) and geranyl tiglate (8.21%) were the major components identified by dynamic headspace using peat (HSD-T), while citronellol (16.88%), geraniol (13.63%), 6,9-guaiadiene (16.98%) and citronellyl formate (6.95%) were identified by dynamic headspace using Porapak Q (HSD-P). Furthermore, this work showed, for the first time, that in natura peat is useful to extract VOCs from leaves of geranium.

White rot fungus Inonotus obliquus pretreatment to improve tran-1,4-polyisoprene extraction and enzymatic saccharification of Eucommia ulmoides leaves.

This study proposes an innovative strategy of lignocellulose biodegradation by Inonotus obliquus under solid-state fermentation in extracting Eucommia ulmoides trans-1,4-polyisoprene (EUG) and producing reducing sugars efficiently. EUG and sugars were obtained through the white rot fungal pretreatment of E. ulmoides leaves, ultrasound-assisted solvent extraction, and enzymatic saccharification. After mere 2-day fermentation, the loss of lignin, cellulose, and hemicelluloses of the leaves achieved 7.11%, 3.47%, and 6.44%, respectively due to the high activity levels of manganese peroxidase (MnP, 973 IU g-1) and lignin peroxidase (LiP, 1341 IU g-1) produced by the fungus. The breakdown of fibrous networks brought higher yields of EUG and reducing sugars. The highest extraction yield of EUG was 4.86% from the 2-day fermented leaves, 31.4% greater than that from the control (3.69%). Meanwhile, the leaf residues after EUG extraction released 97.8 mg g-1 reducing sugars with enzymatic saccharification, 77.5% greater than that from the control (55.1 mg g-1). The results demonstrated that I. obliquus could use E. ulmoides leaves as substrate to produce high-activity-level ligninolytic enzymes in a very short time and the lignocellulose selective degradation of E. ulmoides leaves enhanced the yields of EUG and reducing sugars.

The characteristic smell emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens.

The volatile components emitted from two scale insects, Ceroplastes japonicus and Ceroplastes rubens, were identified using GC-MS analysis. The major volatile components of the solvent extract from C. japonicus were α-humulene (35.8%) and δ-cadinene (17.0%), while those of C. rubens were ß-selinene (10.3%) and ß-elemene (5.1%). In GC/olfactometry, linalool, butyric acid, 3-methylbutyric acid, 2-methylbutyric acid, and vanillin were identified as the odor-active components of the extract from C. japonicus, in addition to trace amounts of trans-4,5-epoxy-(2E)-decenal, 4-methyl-(3E)-hexenoic acid, and phenylacetic acid. With regard to C. rubens, trans-4,5-epoxy-(2E)-decenal, 3-methylbutyric acid, and phenylacetic acid were identified as the odor-active components. Besides, decan-1,4-olide (γ-decalactone) with milky cherry-like note and 3-hydroxy-4,5-dimethylfuran-2(5H)-one (sotolone) with brown sugar-like note were also detected as the characteristic cherry-like sweet-and-sour note of these two scale insects. ABBREVIATIONS: GC: Gas chromatography; GC/O: gas chromatography/olfactometry.

Micro-fabricated packed metal gas preconcentrator for enhanced monitoring of ultralow concentration of isoprene.

A novel non-silicon-based micro-preconcentration device, as a pretreatment component in a portable gas chromatography system, was developed for the preconcentration one of the trace volatile organic compounds (VOCs) in the exhaled gases, which is one typical biomarker for the chronic liver disease (CLD). The device was designed as an array of manifold-shaped rectangular metal micro-channels with flat dimensions of 16 mm × 12.6 mm and the internal empty volume is 14.4 µL on the copper substrate. Instead of the non-silicon fabrication process, the traditional laser etching technology (LET) was optimized to etch micro-channels, and vacuum diffusion welding (VDW) was applied to form internal channels. The fabricated chip was filled with Carbopack X adsorbent. In the testing, the metal gas preconcentrator (MGP) was installed in a commercial GC (gas chromatography) and nitrogen was used as carrier gas and desorbed gas. With the MPG, up to 352 of concentration factor can be achieved for 10 ppb isoprene. The developed MGP, which has advantages of high strength, low cost, good thermal conductivities, can potentially be used for non-invasive screening of advanced liver fibrosis by monitoring isoprene concentrations in exhaled breath.

Hypermonins A and B, two 6-norpolyprenylated acylphloroglucinols with unprecedented skeletons from Hypericum monogynum.

Two new 6-norpolycyclic polyprenylated acylphloroglucinols (PPAPs), hypermonins A (1) and B (2), featuring an undescribed decahydroindeno[1,7-bc]furan ring system, were isolated from the leaves and twigs of Hypericum monogynum. These compounds are a pair of epimers with opposite configurations at the C-5 position. Their structures, including their absolute configurations, were determined by extensive spectroscopic analysis and electronic circular dichroism (ECD) calculations. A plausible biosynthetic pathway of 1 and 2 was also proposed. Compound 1 exhibited a significant protective effect against corticosterone-induced injury in PC12 cells.

Isoprenoids Production from Lipid-Extracted Microalgal Biomass Residues Using Engineered E. coli.

Microalgae are recognized as a third generation feedstock for biofuel production due to their rapid growth rates and lignin-free characteristics. In this study, a lipid extracted microalgal biomass residues was used as the raw material to produce isoprene, α-pinene and ß-pinene with an engineered E. coli strain. We adopted an optimal sulfuric acid hydrolysis method (1:7 ratio of solid to acid solution, 32% (w/v) concentration of sulfuric acid solution at 90 °C for 90 min) to efficiently convert holocellulose into glucose efficiently (6.37 g/L). Futhermore, we explored a novel detoxification strategy (phosphoric acid/calcium hydroxide) to remove inhibitors and notably acetic acid, furfural and 5-hydroxymethylfurfural (5-HMF) were reduced by 5.32%, different number given later 99.19% and 98.22%, respectively. Finally, the fermentation concentrations of isoprene (223.23 mg/L), α-pinene (382.21 µg/L) and ß-pinene (17.4 mg/L) were achieved using the detoxified hydrolysate as the carbon source, equivalent to approximately 86.02%, 90.16% and 88.32% of those produced by the engineered E. coli strain fermented on pure glucose, respectively.

Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm.

Five previously undescribed hemiterpene glycosides, cibotiumbarosides E-I, and two known hemiterpene glucosides, were isolated from the rhizome of Cibotium barometz (L.) J. Sm. The structures of cibotiumbarosides E-I were established by 1D and 2D NMR spectroscopic analyses and HRMS. The absolute configuration of the aglycone of cibotiumbaroside E was assigned by calculated ECD with the TDDFT method. Cibotiumbarosides F and I both exhibited remarkable hepatoprotective activity against APAP-induced acute liver damage in vitro, which were more effective than the positive control, bicyclol. On the other hand, seven hemiterpene glycosides were all inactive in assays of cytotoxicity, neuroprotection, antidiabetes and anti-inflammation.

Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae.

Current studies on microbial isoprene biosynthesis have mostly focused on regulation of the upstream mevalonic acid (MVA) or methyl-erythritol-4-phosphate (MEP) pathway. However, the downstream bottleneck restricting isoprene biosynthesis capacity caused by the weak expression and low activity of plant isoprene synthase (ISPS) under microbial fermentation conditions remains to be alleviated. Here, based on a previously constructed Saccharomyces cerevisiae strain with enhanced precursor supply, we strengthened the downstream pathway through increasing both the expression and activity of ISPS to further improve isoprene production. Firstly, a two-level expression enhancement system was developed for the PGAL1-controlled ISPS by overexpression of GAL 4. Meanwhile, the native GAL1/7/10 promoters were deleted to avoid competition for the transcriptional activator Gal4p, and GAL80 was disrupted to eliminate the dependency of gene expression on galactose induction. The IspS expression was obviously elevated upon enhanced Gal4p supply, and the isoprene production was improved from 6.0mg/L to 23.6mg/L in sealed-vial cultures with sucrose as carbon source. Subsequently, a novel high-throughput screening method was developed based on precursor toxicity and used for ISPS directed evolution towards enhanced catalytic activity. Combinatorial mutagenesis of the resulting ISPS mutants generated the best mutant ISPSM4, introduction of which into the GAL4-overexpressing strain YXM29 achieved 50.2mg/L of isoprene in sealed vials, and the isoprene production reached 640mg/L and 3.7g/L in aerobic batch and fed-batch fermentations, respectively. These results demonstrated the effectiveness of the proposed combinatorial engineering strategy in isoprene biosynthesis, which might also be feasible and instructive for biotechnological production of other valuable chemicals.

Identification and Enrichment of α-Glucosidase-Inhibiting Dihydrostilbene and Flavonoids from Glycyrrhiza uralensis Leaves.

To exploit Glycyrrhiza uralensis resources, we examined the bioactive constituents of G. uralensis leaves. Seven chemical components were isolated by repeat column chromatography, and using spectroscopic methods, their structures were determined to be a novel prenylated dihydrostilbene, α,α'-dihydro-3,5,3',4'-tetrahydroxy-2,5'-diprenylstilbene (1); a methylated flavonoid, quercetin-3-Me ether (4); and 5 prenylated flavonoids: 5'-prenylquercetin (3), 8-[(E)-3-hydroxymethyl-2-butenyl]eriodictyol (7), 6-prenyleriodictyol (5), 5'-prenyleriodictyol (6), and 6-prenylquercetin-3-Me ether (2). Compounds 1-7 and their unprenylated counterparts, glycosides, and other related compounds (8-13) were quantitatively analyzed. Using a macroporous resin column, most of these compounds could be enriched in the 40% to 60% ethanol-eluted fractions. Compounds 1-7 showed strong radical scavenging activity toward DPPH, and most of them demonstrated greater inhibitory activity against α-glucosidase than their unprenylated counterparts.

Polyisoprenylated benzoylphloroglucinol derivatives from Hypericum scabrum.

Four new polyisoprenylated benzoylphloroglucinol derivatives, hyperscabrones J-M (1-4), were isolated from the air-dried aerial parts of Hypericum scabrum. Their structures were elucidated by spectroscopic methods and were subsequently confirmed by comparing with data of known compounds. The absolute configuration of the bicyclo[3.3.1]nonane-2,4,9-trione core was defined by the experimental and calculated electronic circular dichroism (ECD) spectra. The evaluation of their hepatoprotective activities against paracetamol-induced HepG2 cell damage showed that compounds 2 and 4 exhibited significant hepatoprotection at 10µM.

Antibacterial activities of the methanol extracts, fractions and compounds from Harungana madagascariensis Lam. ex Poir. (Hypericaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Harungana madagascariensis Lam. ex Poir. (Hypericaceae) is used in folk medicine to treat a variety of human ailments, mainly antibacterial, antifungal, antiviral and viral infections. In the present study, the methanol extract from the leaves (HML) and bark (HMB) of this plant as well as fractions (HMBa-c), sub-fractions (HMBa1-5) and compounds isolated from HMBa and HMBb namely betulinic acid (1), madagascin (2), ferruginin A (3) and Kaempferol-3-O-ß-d-glucopyranoside (4) were tested for their antimicrobial activities against a panel of 28 g-negative bacteria including multidrug resistant (MDR) phenotypes. MATERIALS AND METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the above samples; column chromatography was used for the fractionation and purification of the bark extract whilst the chemical structures of compounds were determined using spectroscopic techniques. RESULTS: Crude extract HMB together with fraction HMBa and sub-fraction HMBa3 were active on the 28 tested bacterial strains. HML as well as fractions HMBb, HMBc and sub-fractions HMBa1, HMBa2, HMBa4 and HMBa5 were selectively active. MIC values below or equal to 1024µg/mL were recorded with these samples on 92.9% (for HML and HMBa 4), 82.1% (for HMBb), 78.6% (for HMBa2), 50.0% (for HMBa5) and 42.9% (for HMBc) tested bacteria. For crude material, the lowest MIC value below 8µg/mL was obtained with HMB against Escherichia coli ATCC10536 and W3110 strains, and with sub-fraction HMBa3 against Klebsiella pneumoniae K2 strains. MIC values below 10µg/mL were recorded with compound 3 against E. coli ATCC10536, Enterobacter aerogenes ATCC13048 and EA294, Pseudomonas aeruginosa PA01, K. pneumoniae K2 and Kp55 and Enterobacter cloacae BM67. CONCLUSIONS: Harungana madagascariensis is a potential source of antimicrobial drugs to fight against MDR bacteria. The anthranol 3 is the main antibacterial constituents of the bark of the plant. HMB and compound 3 deserve further investigations to develop natural drug to combat Gram-negative bacteria and otherwise MDR phenotypes.

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli.

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The (13)C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0g/L isoprene with a yield of 0.267g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.

Phenolic constituents of the Bangladeshi medicinal plant Pothos scandens and their anti-estrogenic, hyaluronidase inhibition, and histamine release inhibitory activities.

Extracts from the stem and roots of the Bangladeshi medicinal plant Pothos scandens L. (Araceae) were isolated, and three hemiterpene glucoside aromatic esters, pothobanosides A (1), B (2), and C (3), and a phenylisobutanoid, pothobanol (4), along with 14 known compounds, were characterized. The isolates were tested for their estrogenic/anti-estrogenic activity using the estrogen-responsive human breast cancer cell lines MCF-7 and T47D, and syringoyl derivatives (2, 3, and canthoside B) showed strong inhibitory activity against both cell lines. Their less oxygenated analogs (1, and markhamioside F) were almost inactive. The isolates were also evaluated for hyaluronidase and histamine release inhibitory activities, and pothobanoside A (1) showed significant hyaluronidase inhibitory activity among the isolated compounds, which was similar to that of the positive control rosmarinic acid. Because hyaluronidase produces an angiogenic response that has been implicated in tumor invasiveness and metastasis, 1 could be valuable as an anti-tumor compound with a different mechanism of action from related compounds (2, 3). Pothobanoside C (3) and pothobanol (4) were also found to inhibit histamine release to a similar degree to the positive control epigallocatechin 3-O-(3"-O-methyl)-gallate. The histamine release inhibitory potency of these isolates may support the traditional uses of this plant in folk medicine.

Novel chromenedione derivatives displaying inhibition of protein tyrosine phosphatase 1B (PTP1B) from Flemingia philippinensis.

Protein tyrosine phosphatase 1B (PTP1B) is an important target to treat obesity and diabetes due to its key roles in insulin and leptin signaling. The MeOH extracts of the root bark of Flemingia philippinensis yielded eight inhibitory molecules (1-8) capable of targeting PTP1B. Three of them were identified to be novel compounds, philippin A (1), philippin B (2), and philippin C (3) which have a rare 3-phenylpropanoyl chromenedione skeleton. The other compounds (4-8) were known prenylated isoflavones. All compounds (1-8) inhibited PTP1B in a dose dependent manner with IC50s ranging between 2.4 and 29.4µM. The most potent compound emerged to be prenylated isoflavone 5 (IC50=2.4µM). In kinetic studies, chromenedione derivatives (1-3) emerged to be reversible, competitive inhibitors, whereas prenylated isoflavones (5-8) were noncompetitive inhibitors.

Polyphenols isolated from Broussonetia kazinoki prevent cytokine-induced ß-cell damage and the development of type 1 diabetes.

The axis of nuclear factor κB (NF-κB)-inducible NO synthase (iNOS)-nitric oxide plays a key role in cytokine- and streptozotocin-mediated pancreatic ß-cell damage. In this study, we investigated the effects of kazinol C and isokazinol D isolated from Broussonetia kazinoki on the ß-cell viability and function. RINm5F cells and primary islets were used for in vitro and ex vivo cytokine toxicity experiments, respectively. For type 1 diabetes induction, mice were injected with multiple low-dose streptozotocin (MLDS). Cytokine-induced toxicity was completely abolished in both RINm5F cells and islets that were pretreated with either kazinol C or isokazinol D. Both kazinols inhibited the NF-κB signaling pathway, thereby inhibiting cytokine-mediated iNOS induction, nitric oxide production, apoptotic cell death and defects in insulin secretion. Moreover, the occurrence of diabetes in MLDS-treated mice was efficiently attenuated in kazinol-pretreated mice. Immunohistochemical analysis revealed that the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive apoptotic cells and nuclear p65-positive cells were significantly decreased in kazinol-pretreated mice. Our results suggest that kazinol C and isokazinol D block the NF-κB pathway, thus reducing the extent of ß-cell damage. Therefore, kazinol C and isokazinol D may have therapeutic value in delaying pancreatic ß-cell damage in type 1 diabetes.

The diversion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate from the 2-C-methyl-D-erythritol 4-phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana.

2-C-Methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) is an intermediate of the plastid-localized 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co-factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds-3 mutant, defective in the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2-C-methyl-D-erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds-3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds-3 mutant also showed enhanced resistance to the phloem-feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP-mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds-3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.

A new hemiterpene glycoside from the ripe tomatoes.

A new hemiterpene glycoside (1) was isolated from ripe tomatoes (the fruit of Lycopersicon esculentum, Solanaceae) along with eight known compounds. The chemical structure of 1 was determined to be 2-methylbutan-1-ol ß-d-glucopyranosyl-(1 → 6)-ß-d-glucopyranoside, based on spectroscopic data as well as chemical evidence. In addition, the radical-scavenging activities of the isolated compounds on the free radical of 1,1-diphenyl-2-picrylhydrazyl were examined. Among the tested compounds, tryptophan, 4-O-ß-d-glucopyranosyl caffeic acid and dihydro-p-coumaryl alcohol γ-O-ß-d-glucopyranoside demonstrated 42.0%, 50.1% and 76.0% scavenging activities, respectively, at a concentration of 0.5 mM.

Screening for novel plant sources of prenyloxyanthraquinones: Senna alexandrina Mill. and Aloe vera (L.) Burm. F.

As a continuation of our ongoing studies aimed to reveal the presence of oxyprenylated anthraquinones in plants claimed to have a laxative effect, in this article, we describe the extraction and HPLC separation of madagascin (3-isopentenyloxyemodin) and 3-geranyloxyemodine from dried leaves and fruits of Senna alexandrina Mill. (Leguminosae) and leaves and gel of Aloe vera (L.) Burm. F. (Xanthorrhoeaceae). Both compounds are described herein for the first time as components of extracts of the title plants.