MS diagnostic model and rapid distinguishing of bioactive limonoids in fruits of Melia toosendan using solid-phase extraction coupled with LC-MS/MS.

INTRODUCTION: Melia toosendan Sieb. et Zucc. has been used as a Chinese folk medicine for roundworm treatment since ancient times. Many diverse limonoids have been isolated from Meliaceae plants, but it remains difficult to isolate and identify other limonoids because of their small natural concentrations. OBJECTIVE: This study was performed to overcome the difficulties associated with fast and accurate identification of limonoids and establish a reliable and sensitive method for the analysis of minor limonoids in M. toosendan fruits. METHODS: An efficient strategy for enrichment, detection, and identification of minor limonoids from M. toosendan fruits using solid-phase extraction with high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (SPE-HPLC-Q-TOF-MS/MS) was developed herein. RESULTS: Characteristic fragmentations and fragmentation ions containing trichilin-, nimbin-, and vilasinin-class limonoid skeletons were initially studied, and characteristic diagnostic ions involved retro Diels-Alder (RDA) reactions or homolytic cleavages, which were used to identify minor limonoids. In total, 13 limonoids, including four new ones, were identified. CONCLUSION: This is the first report on the analysis of M. toosendan fruits to identify limonoids. This novel analysis method may stimulate further research regarding the identification of limonoids in other plant species.

Trichiconlides A and B: two novel limonoids from the fruits of Trichilia connaroides.

Trichiconlides A (1) and B (2), two novel limonoids, were isolated from the fruits of Trichilia connaroides. Compound 1 is a novel limonoid with a highly rearranged A/B ring system and an intact D ring, which formed an unprecedented 5/6/5/6/5 carbon skeleton. Trichiconlide B (2) possesses a 10-oxatricyclo[3.3.1.(3,8)]decane core and formed a cage-like structure located between rings A and C. Their structures and absolute configurations were elucidated by spectroscopic methods, single crystal X-ray diffraction, the ECD exciton chirality method and TDDFT/ECD calculations. 1 showed a moderate inhibitory effect on nitric oxide (NO) production.

Four previously undescribed diketopiperazines from marine fungus Aspergillus puniceus FAHY0085 and their effects on liver X receptor α.

Four previously undescribed diketopiperazine-type alkaloids including one oxepin-containing diketopiperazine-type alkaloid, oxepinamide L (1), three 4-quinazolinone alkaloids, puniceloids E-G (10-12), together with 12 known analogues, protuboxepin D (2), oxepinamides D-G, J-K and I (3-9), puniceloids B-D (13-15) and protubonine B (16), were isolated from the culture of the marine-derived fungus Aspergillus puniceus FAHY0085. The structures of the previously undescribed compounds were comprehensively elucidated by detailed interpretation of their NMR and HRESIMS data. Their absolute configurations were unambiguously determined by ROESY experiments, Marfey's method, calculated ECD experiments and single-crystal X-ray diffraction analysis. Compounds (3-4, 6-8, 14-15) were evaluated for their cytotoxic activity against HepG2, MCF-7, SW1116 and HeLa cells and compound 6 and 14 showed moderate cytotoxic activity against HeLa cells with IC50 49.61 ± 2.91 and 28.38 ± 1.57 µM, respectively. Compounds (1-8, 11-15) were screened for their transcriptional activation of liver X receptor α and compound 11 with known compounds 13-15 showed significant transcriptional activation of liver X receptor α with EC50 values in the range 2-50 µM.

Efficient ilamycins production utilizing Enteromorpha prolifera by metabolically engineered Streptomyces atratus.

With the invasion of green tides and the increase of urban green areas worldwide, multimillion tons of Enteromorpha need to be reutilized. In this study, Enteromorpha prolifera powder is considered a promising biomass resource for the production of commercial chemical products production. Ilamycins, novel cyclic heptapeptides with significant anti-TB activities, are isolated from Streptomyces atratus SCSIO ZH16, a deep-sea-derived strain. Using EP powder as a nitrogen source, the production of ilamycins reached 709.97 mg/L through optimization of the nitrogen source using the engineered strain S. atratus SCSIO ZH16 ΔR. After mutant strain constructions and tests, strain S. atratus SCSIO ZH16 ΔR::bldD EP powder achieved a higher production titer of ilamycins. Furthermore, the production titer of ilamycins and ilamycin E reached 1561.77 mg/L and 745.44 mg/L, respectively, in a 5 L bioreactor. This study suggests that E. prolifera is a promising and eco-friendly nitrogen source for the production of ilamycins.

Development of a High-Titer Culture Medium for the Production of Cholesterol by Engineered Saccharomyces cerevisiae and Its Fed-Batch Cultivation Strategy.

Steroids are a class of compounds with cyclopentane polyhydrophenanthrene as the parent nucleus, and they usually have unique biological and pharmacological activities. Most of the biosynthesis of steroids is completed by a series of enzymatic reactions starting from cholesterol. Synthetic biology can be used to synthesize cholesterol in engineered microorganisms, but the production of cholesterol is too low to further produce other high-value steroids from cholesterol as the raw material and precursor. In this work, combinational strategies were established to increase the production of cholesterol in engineered Saccharomyces cerevisiae RH6829. The basic medium for high cholesterol production was selected by screening 8 kinds of culture media. Single-factor optimization of the carbon and nitrogen sources of the culture medium, and the addition of calcium ions, zinc ions and citric acid, further increased the cholesterol production to 192.53 mg/l. In the 5-L bioreactor, through the establishment of strategies for glucose and citric acid feeding and dissolved oxygen regulation, the cholesterol production was further increased to 339.87 mg/l, which was 734% higher than that in the original medium. This is the highest titer of cholesterol produced by microorganisms currently reported. The fermentation program has also been conducted in a 50-L bioreactor to prove its stability and feasibility.

Further new nardosinane-type sesquiterpenoids from the Xisha soft coral Litophyton nigrum.

Further chemical investigation of the Xisha soft coral Litophyton nigrum has resulted in the isolation of four new nardosinane-type sesquiterpenoids, namely linardosinenes D-G (1-4). The structures of new compounds were elucidated by extensive analyses of their spectroscopic data and by comparison with the reported data of known related ones. All compounds exhibited weak inhibitory effect against bromodomain-containing protein 4 (BRD4), a promising therapeutic target in various human diseases, at a concentration of 10 µM.

Improvement of Curvulamine Production by Precursors Co-addition Strategy in Liquid Culture of Marine-Derived Fungus Curvularia sp. IFB-Z10.

Curvulamine, a novel scaffold alkaloid with remarkable selective antibacterial activity, is produced by marine fungus Curvularia sp. IFB-Z10. However, its deep pharmaceutical research and application are severely restricted by the low yield, which needs to be solved urgently. The purpose of this study was to improve curvulamine production via precursors co-addition strategy and further reveal the regulation mechanism. In this work, the optimal precursors co-addition conditions were firstly obtained, and curvulamine production achieved 166.74 mg/L with the supply of 250 mg/L alanine and 200 mg/L proline at 60 h, which was 4.08 times that of control. It was observed that under alanine and proline stimulation, fungus exhibited the morphology of a small-diameter compact pellet. Furthermore, the organic acid levels in central carbon metabolism (CCM) were declined with precursors supplement. Besides, precursors also induced the critical biosynthetic gene transcriptions. The above findings collectively promoted curvulamine synthesis. Finally, Curvularia sp. IFB-Z10 fermentation process was successfully established by feeding alanine and proline at 0.021 g/L/h and 0.017 g/L/h rate from 60 to 72 h, and curvulamine production reached 133.58 mg/L in a 5-L bioreactor. The information acquired would facilitate the enhancement of curvulamine yield in submerged fermentation and the research on synthesis regulation of other alkaloids.

Highly oxygenous trichilin-type limonoids from Trichilia sinensis.

Six new trichilin-type limonoids (1-6) with C-19/29 lactol or acetal bridge and a new ring intact limonoid (7) were isolated from the desiccative ripe fruits of Trichilia sinensis. Their structures were determined by extensive spectroscopic methods including 1H NMR, 13C NMR, HSQC, HMBC, ROESY experiments as well as HRESI-MS data. All isolated compounds were evaluated for toxicities against human pulmonary carcinoma A549 and Hela cell lines by sulforhodamine B (SRB) method. Compound 7 showed weak inhibitory activity in Hela cell line at 40 µmol·L-1.

Ammonium Acetate Supplement Strategy for Enhancement of Chaetominine Production in Liquid Culture of Marine-Derived Aspergillus fumigatus CY018.

Pharmacological research on (CHA), a marine-derived quinazolinone alkaloid with significant cytotoxic activity, is restricted by low yields and is a problem that needs to be settled urgently. In this work, the selection of additional nitrogen sources and the optimization of additional concentrations and longer fermentation times using ammonium acetate, were investigated. CHA production was optimized to 62.1 mg/l with the addition of 50 mM ammonium acetate at 120 h of the fermentation in the shaker flask. This feeding strategy significantly increased 3- deoxy-arabino-heptulosonate-7-phosphate synthase activity and transcript levels of critical genes (laeA, dahp and trpC) in the shikimate pathway compared with the non-treatment group. In addition, the selection of the feeding rate (0.01 and 0.03 g/l/h) was investigated in a 5-L bioreactor. As a result, CHA production was increased by 57.9 mg/l with a 0.01 g/l/h ammonium acetate feeding rate. This work shows that the strategy of ammonium acetate supplementation had an effective role in improving CHA production by Aspergillus fumigatus CY018. It also shows that this strategy could serve as an important example of large-scale fermentation of a marine fungus in submerged culture.

Curvulaide A, a bicyclic polyketide with anti-anaerobic bacteria activity from marine-derived Curvularia sp.

Curvulaide A (1), a new bicyclic polyketide, together with its known congener preussilide D, were produced by solid-state fermentation with Curvularia sp. IFB-Z10. The structure of 1 was elucidated using a combination of spectral methods, including extensive one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data, high resolution electrospray ionization mass spectrometry (HRESIMS), experimental and calculated electronic circular dichroism (ECD) spectra. Curvulaide A (1) exhibited moderate anti-anaerobic bacteria activity against periodontal pathogen Porphyromonas gingivalis with the MIC value of 62.5 µM, and showed moderate cytotoxicity against human hepatoma cell lines BEL7402/5-Fu with IC50 values of 12.46 µM.

Stimulatory Effects of Sugarcane Molasses on Fumigaclavine C Biosynthesis by Aspergillus fumigatus CY018 via Biofilm Enhancement.

Biofilms are of vital significance in bioconversion and biotechnological processes. In this work, sugarcane molasses was used to enhance biofilms for the improvement of the production of fumigaclavine C (FC), a conidiation-associated ergot alkaloid with strong anti-inflammatory activities. Biofilm formation was more greatly induced by the addition of molasses than the addition of other reported biofilm inducers. With the optimal molasses concentration (400 g/l), the biofilm biomass was 6-fold higher than that with sucrose, and FC and conidia production was increased by 5.8- and 3.1-fold, respectively. Moreover, the global secondary metabolism regulatory gene laeA, FC biosynthetic gene fgaOx3, and asexual central regulatory genes brlA and wetA were upregulated in molasses-based biofilms, suggesting the upregulation of both asexual development and FC biosynthesis. This study provides novel insight into the stimulatory effects of molasses on biofilm formation and supports the widespread application of molasses as an inexpensive raw material and effective inducer for biofilm production.

Co-addition Strategy for Enhancement of Chaetominine from Submerged Fermentation of Aspergillus fumigatus CY018.

Chaetominine (CHA), a novel framework tripeptide alkaloid, imparts an attractive cytotoxic against the human leukemia cell line K562, which is produced by Aspergillus fumigatus CY018. However, its pharmacological research is restricted by low yields in submerged culture, which needs to be resolved immediately by biotechnology. In this work, a co-addition strategy was applied to promote CHA production based on related inhibitors' addition and precursors' addition, inspired by the biosynthetic pathway analysis of CHA. CHA production reached 53.87 mg/L by addition of 10 mM shikimate, 10 mM anthranilate, 20 mM tryptophan, and 10 mM alanine in shake flask. Compared to the control without addition of precursors, the activity of 3-deoxy-arabino-heptulosonate-7-phospahte (DAHP) synthase was significantly improved and the transcription levels of critical genes in shikimate pathway were up-regulated responded to the co-addition of precursors. The improvement of CHA production by co-addition of precursors was also successfully reproduced in the lab-scale bioreactor (5-L) system, in which CHA production reached 46.10 mg/L. This work demonstrated that precursors' co-addition was an effective strategy for increasing CHA production, and the information obtained might be useful to the further improvement of CHA on a large scale.

Trichiconlides CF, four new limonoids with 1,2-seco phragmalin-type carbon skeleton from the fruits of Trichilia connaroides.

Phytochemical investigation of the fruits from Trichilia connaroides led to the isolation of four rare 1,2-seco phragmalin-type limonoids (1-4) with C-7/28 δ-lactone ring along with two known triterpenoids (5-6). An oxygen bridge between C-1 and C-2 formed an rare 9-oxa-tricyclo[3.3.2.17,10]undecane-2-ene moiety in structure of 1 and 2, and 3-4 possessed a bicyclo[5.2.14,10]decane skeleton. Their structures were established based on extensive spectroscopic methods and their absolute configurations of 1 were confirmed by TDDFT/ECD calculation and comparative analysis. Compounds 5-6 showed moderate inhibitory effects on Hela tumor cell lines with inhibition ratio values of 83.8% and 87.1% at concentration 40µM, respectively.

Trijugin- and mexicanolide-type limonoids from the fruits of Heynea trijuga that reverse multidrug resistance in MCF-7/DOX cells.

Eleven previously undescribed limonoids, trichisins A-K, including eight structural analogues A-H of trijugin and three H-J mexicanolide derivatives, together with two known mexicanolide derivatives were isolated from the fruits of Heynea trijuga Roxb. ex Sims. The structure determination was based on extensive physical data analyses (NMR, MS), and their basic skeletons and the absolute configurations of trichisins A, B, E, K and trichiconnarone A were assigned via X-ray crystallographic analysis (Cu Kα radiation). The hemiketal motifs in trijugins A, B, and E-G are rare in limonoids. Bioactivity screenings suggested that the trijugin H and mexicanolide-type trichiconnarones A and B limonoids were effective in reversing resistance in MCF-7/DOX cells at a nontoxic concentration of 50 µM with IC50 values of 12.45, 10.86, and 14.96 µM, respectively.

Cipadessains A-K, eleven limonoids from the fruits of Cipadessa cinerascens.

Eleven new mexicanolide-type limonoids, cipadessains A-K (1-11), were isolated from the fruits of Cipadessa cinerascens (Pellegr) Hand.-Mazz. Their planar structures were determined based on IR, UV, 1D and 2D NMR spectra and HRESIMS data. The absolute configuration of 1 was elucidated by single-crystal X-ray diffraction using mirror Cu Kα radiation, and that of compounds 2-8 were determined by ECD analysis. Two mexicanolides bearing methoxybutenolide moiety originated from the furan ring 3 and 6, showed significant cytotoxicity against HepG2 cell line with IC50 values of 5.23 ± 0.12, 8.67 ± 1.02 µM, respectively; and NO inhibitory activities in LPS-activated RAW 264.7 macrophages at nontoxic concentration (IC50 5.79 ± 0.18, 6.93 ± 0.89 µM, respectively).

Diverse tritepenoids from the fruits of Walsura robusta and their reversal of multidrug resistance phenotype in human breast cancer cells.

Four 18 (13 â†’ 14)-abeo limonoids, five cedrelone limonoids, and five walsurin limonoids were isolated from the fruits of Walsura robusta together with 21 known compounds. Their structures were determined by extensive studies of their one- and two-dimensional NMR spectra and mass spectroscopy results. Among all the isolated compounds, the absolute configurations of 11-oxo-dihydrocedrelone, walsuronoid B, dysoxylumosin G, and walsunoid H were confirmed by single crystal X-ray diffraction. Selected compounds were evaluated for their reversal of multidrug resistance (MDR) phenotype in human breast cancer cells in vitro. Walsurin A showed significant effect in sensitization of these resistant cancer cells at non-toxic concentration to doxorubicin with the modulation factor of 62.76.

Walrobsins A and B, Two Anti-inflammatory Limonoids from Root Barks of Walsura robusta.

Walrobsins A (1) and B (2), two limonoids featuring an unprecedented 5-oxatricyclo[5.4.11,4]hendecane ring system, were isolated from the root barks of Walsura robusta. Their structures, including their absolute configurations, were determined by analyses of HR-ESIMS, 1D/2D NMR, and X-ray crystallography. Compounds 1 and 2 possessed a stable hemiketal structure formed between the OH-11 and 3-carbonyl group in the hexatomic oxoheterocyclic ring. Compound 1 showed significant anti-inflammatory activity with an IC50 value of 7.8 µM and inhibited the expression of iNOS and IL-1ß in a dose-dependent manner.

Icariside II, a natural mTOR inhibitor, disrupts aberrant energy homeostasis via suppressing mTORC1-4E-BP1 axis in sarcoma cells.

The aberrant energy homeostasis that characterized by high rate of energy production (glycolysis) and energy consumption (mRNA translation) is associated with the development of cancer. As mammalian target of rapamycin (mTOR) is a critical regulator of aberrant energy homeostasis, it is an attractive target for anti-tumor intervention. The flavonoid compound Icariside II (IS) is a natural mTOR inhibitor derived from Epimedium. Koreanum. Herein, we evaluate the effect of IS on aberrant energy homeostasis. The reduction of glycolysis and mRNA translation in U2OS (osteosarcoma), S180 (fibrosarcoma) and SW1535 (chondrosarcoma) cells observed in our study, indicate that, IS inhibits aberrant energy homeostasis. This inhibition is found to be due to suppression of mammalian target of rapamycin complex 1 (mTORC1)-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) axis through blocking the assembly of mTORC1. Furthermore, IS inhibits the cap-dependent translation of c-myc through mTORC1-4E-BP1 axis which links the relationship between mRNA translation and glycolysis. Inhibition of aberrant energy homeostasis by IS, contributes to its in vitro and in vivo anti-proliferation activity. These data indicate that IS disrupts aberrant energy homeostasis of sarcoma cells through suppression of mTORC1-4E-BP1 axis, providing a novel mechanism of IS to inhibit cell proliferation in sarcoma cells.

Cytotoxic Rocaglate Derivatives from Leaves of Aglaia perviridis.

Rocaglates are a series of structurally complex secondary metabolites with considerable cytotoxicity that have been isolated from plants of the Aglaia genus (Meliaceae). A new rocaglate (aglapervirisin A, 1) and its eight new biosynthetic precursors of rocaglate (aglapervirisins B-J, 2-9) together with five known compounds, were isolated from the leaves of Aglaia perviridis. Their structures were elucidated based on a joint effort of spectroscopic methods [IR, UV, MS, ECD, 1D- and 2D-NMR, HRESIMS], chemical conversion and single-crystal X-ray diffraction. Among these isolates, three (1, 10-11) were silvestrols, a rare subtype rocaglates, exhibiting notable cytotoxicity against four human tumor cell lines, with IC50 values between 8.0 and 15.0 nM. Aglapervirisin A (1) induces cell cycle arrest at the G2/M-phase boundary at concentration 10 nM accompanied by reductions in the expression levels of Cdc2 and Cdc25C in HepG2 cells after 72h co-incubation, and further induces the apoptosis of HepG2 cells at concentrations over 160 nM.