Identification and Biological Activities of Long‐Chain Peptaibols Produced by a Marine‐Derived Strain of Trichoderma longibrachiatum

Six long‐chain peptaibols, 1  –  6 , were identified from agar cultures of a marine‐derived Trichoderma longibrachiatum Rifai strain (MMS151) isolated from blue mussels. The structure elucidation was carried out using electrospray ionization ion trap mass spectrometry (ESI‐IT‐MS) and GC/EI‐MS. The long‐chain peptaibols exhibited the general building scheme Ac‐Aib‐Ala‐Aib‐Ala‐Aib‐XXX‐Gln‐Aib‐Vxx‐Aib‐Gly‐XXX‐Aib‐Pro‐Vxx‐Aib‐XXX‐Gln‐Gln‐Pheol and were similar or identical to recurrent 20‐residue peptaibols produced by Trichoderma spp. Three new sequences were identified and were called longibrachins A‐0, A‐II‐a, and A‐IV‐b. The isolated peptaibols were assayed for cytotoxic, antibacterial, and antifungal activities, and acute toxicity on Dipteran larvae.     

Caryophyllene Sesquiterpenes from Pulicaria vulgaris Gaertn.: Isolation,Structure Determination,Bioactivity and Structure−Activity Relationship

A new caryophyllene, named pulicaryenne A ( 1 ), along with four other known caryophyllene derivatives ( 2 , 3 , 4 and 5 ) were isolated from the ethyl acetate extract of aerial parts of Pulicaria vulgaris Gaertn . (Asteraceae). All compounds were isolated for the first time from this species. Compound 2 was identified as a new epimer of a known caryophyllene derivative isolated previously from P. dysenterica. Their structures were established by spectroscopic means including NMR analysis (1D‐ and 2D‐NMR) and ESI‐TOF‐MS. All compounds were evaluated for their anticholinesterase, antityrosinase and cytotoxic activities against two human cell lines (A549 and HeLa). Results showed that compound 5 exhibited the highest cytotoxic effect against A549 and anticholinesterase activity with IC₅₀ values of 8.50±0.75 and 6.45±0.09 μg/mL, respectively. Furthermore, compound 5 showed also an interesting antityrosinase activity with percent inhibition value of 79.0±2.5 % at 50 μg/mL. The bioactivity and drug likeness scores of the isolated compounds 1–5 were calculated using Molinspiration software and discussed. These results may suggest that the five caryophyllene derivatives endowed with good biological properties, which could be used as bioactive alternatives in pharmaceutical preparations.     

Penicamedine A,a Highly Oxygenated Hexacyclic Indole Alkaloid from Penicillium camemberti

A highly oxygenated hexacyclic indole alkaloid, penicamedine A ( 1 ), bearing a rare furan ring, was isolated from the culture broth of Penicillium camemberti, together with two known analogs, iso‐α‐cyclopiazonic acid ( 2 ) and cyclopiazonic acid ( 3 ). The structure of 1 was elucidated by comprehensive spectroscopic analyses including NMR and HR‐ESI‐MS. Its absolute configuration was further confirmed unambiguously by single‐crystal X‐ray diffraction analysis. Compound 1 was evaluated for anti‐HIV activity with p24 assays and tested for cytotoxic activities against five human cancer cell lines, including HL‐60, SMMC‐7721, A‐549, MCF‐7, SW480, and the immortalized non‐cancerous human pulmonary epithelial cell line BEAS‐2B by MTS method.     

Methionine sulfoximine supplementation enhances productivity in GS–CHOK1SV cell lines through glutathione biosynthesis

In Lonza Biologics' GS Gene Expression System?, recombinant protein‐producing GS–CHOK1SV cell lines are generated by transfection with an expression vector encoding both GS and the protein product genes followed by selection in MSX and glutamine‐free medium. MSX is required to inhibit endogenous CHOK1SV GS, and in effect create a glutamine auxotrophy in the host that can be complemented by the expression vector encoded GS in selected cell lines. However, MSX is not a specific inhibitor of GS as it also inhibits the activity of GCL (a key enzyme in the glutathione biosynthesis pathway) to a similar extent. Glutathione species (GSH and GSSG) have been shown to provide both oxidizing and reducing equivalents to ER‐resident oxidoreductases, raising the possibility that selection for transfectants with increased GCL expression could result in the isolation of GS–CHOKISV cell lines with improved capacity for recombinant protein production. In this study we have begun to address the relationship between MSX supplementation, the amount of intracellular GCL subunit and mAb production from a panel of GS–CHOK1SV cell lines. We then evaluated the influence of reduced GCL activity on batch culture of an industrially relevant mAb‐producing GS–CHOK1SV cell line. To the best of our knowledge, this paper describes for the first time the change in expression of GCL subunits and recombinant mAb production in these cell lines with the degree of MSX supplementation in routine subculture. Our data also shows that partial inhibition of GCL activity in medium containing 75 µM MSX increases mAb productivity, and its more specific inhibitor BSO used at a concentration of 80 µM in medium increases the specific rate of mAb production eight‐fold and the concentration in harvest medium by two‐fold. These findings support a link between the inhibition of glutathione biosynthesis and recombinant protein production in industrially relevant systems and provide a process‐driven method for increasing mAb productivity from GS–CHOK1SV cell lines. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:17–25, 2017     

Three New Triterpenoid Saponins from Notoginseng Medicinal Fungal Substance

Three new triterpenoid saponins, named ginsenoside‐Rh₂₃ ( 1 ), ginsenoside‐Rh₂₄ ( 2 ), and ginsenoside‐Rh₂₅ ( 3 ), were isolated from notoginseng medicinal fungal substance. Their structures were elucidated by a combination of 1D‐ and 2D‐NMR, MS and chemical analysis. Compounds 1  –  3 exhibited moderate cytotoxic activity against MCF‐7 and NCI‐H460 cancer cell lines.     

New Trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.     

6,7-seco-ent-kaurane diterpenoids from Isodon sculponeatus with cytotoxic activity

Six new 6,7‐seco‐ent‐kaurane diterpenoids, sculponeatins N–S ( 1 – 6 , resp.), together with eleven known analogues, 7 – 17 , were isolated from the aerial parts of Isodon sculponeatus. The structures of compounds 1 – 6 were elucidated by spectroscopic methods including extensive 1D‐ and 2D‐NMR experiments, as well as HR‐ESI‐MS analysis. All diterpenoids obtained were assayed for their cytotoxic activity against K562 and HepG2 human tumor cell lines. Among them, compound 1 showed the most significant cytotoxicity with the IC₅₀ values of 0.21 and 0.29 μM , respectively. The structure–activity relationships are discussed.     

Sinuladiterpenes A–F,New Cembrane Diterpenes from Sinularia flexibilis

Chromatographic investigation of the octocoral Sinularia flexibilis afforded six new cembrane diterpenes, sinuladiterpenes A–F ( 1 – 6 , resp.), in addition to four known cembranolides, 11‐episinulariolide acetate, 11 ‐ dehydrosinulariolide, 11‐episinulariolide, and sinulariolide. Their structures were elucidated by spectroscopic analysis, especially 2D‐NMR and HR‐ESI‐MS. Compound 2 exhibited significant in vitro cytotoxic activity against human colon adenocarcinoma (WiDr) cell line.     

Caesalpanins A–C,Three Dimeric Cassane Diterpenoids from the Seeds of Caesalpinia sappan L.

Three dimeric cassane diterpenoids, caesalpanins A–C, were isolated from the seeds of Caesalpinia sappan L., as well as three known compounds. Their structures were determined via analysis of 1D‐, 2D‐NMR, and HR‐ESI‐MS data. Caesalpanins A and B were the second and third compounds that presented a nitrogen‐containing cassane diterpenoid dimer linked through one ether bond between C‐19 and C‐20′. Caesalpanin B exhibited moderate cytotoxic activity against MCF‐7 cell lines with IC₅₀ value of 29.98 μm . Caesalpanins A and B had weak inhibitory effects against LPS‐induced nitric oxide (NO) production in RAW 264.7 macrophages at 50 μm with inhibitory rate of 36.01 % and 32.93 %, respectively.     

Cytotoxic Spliceostatin Analogs from Pseudomonas sp.

Two new spliceostatin analogs, designed as spliceostatins J and K ( 1 and 2 ), were isolated and identified from the culture of Pseudomonas sp., along with two known ones, FR901464 ( 3 ) and spliceostatin E ( 4 ). Their structures were elucidated by detailed interpretation of their spectroscopic data, especially 2D‐NMR and HR‐ESI‐MS. Spliceostatin J ( 1 ) represented the first example of spliceostatins bearing an unusual hexahydrofuro[3,4‐b]furan moiety. Biological assay showed all the isolated compounds except 1 displayed potent cytotoxic activities against two cancer cell lines (MDA‐MB‐231 and A‐549). Structure‐activity‐relationship studies revealed that the tetrahydropyran ring in spliceostatin analogs was necessary for their bioactive retention.     

Isocostic Acid,a Promising Bioactive Agent from the Essential Oil of Inula viscosa (L.): Insights from Drug Likeness Properties,Molecular Docking and SAR Analysis

The chemical composition of the essential oil (LEO) and its volatile fractions (V₁–V₁₀) collected during the hydrodistillation process every 15 min from the fresh leaves of I. viscosa (L.), growing in Tunisia, were analyzed by GC‐FID and GC/MS. Eighty‐two compounds, representing 90.9–99.4 % of the total samples, were identified. The crude essential oil (LEO) and its fractions (V₁–V₁₀) were characterized by the presence of a high amount of oxygenated sesquiterpenes (82.7–95.8 %). Isocostic acid ( 1 ) was found to be the most abundant component (37.4–83.9 %) and was isolated from the same essential oil over silica gel column chromatography and identified by spectroscopic methods (¹H, ¹³C, DEPT 135 NMR and EI‐MS) and by comparison with literature data. Furthermore, the fresh leaves essential oil (LEO), its volatile fractions (V₁–V₁₀) as well as compound 1 were screened for their antibacterial, antityrosinase, anticholinesterase and anti‐5‐lipoxygenase activities. It was found that the isolated compound 1 exhibited an interesting antibacterial activity against Staphylococcus aureus ATCC 25923 (MIC=32 μg/mL) and Enterococcus faecalis ATCC 29212 (MIC=32 μg/mL) and the highest antityrosinase activity (IC₅₀=13.82±0.87 μg/mL). Compound 1 was also found to be able to strongly inhibit 5‐lipoxygenase with an IC₅₀ value of 59.21±0.85 μg/mL. The bioactivity and drug likeness scores of compound 1 were calculated using Molinspiration software and interpreted, and the structure‐activity relationship (SAR) was discussed with the help of molecular docking analysis.     

Chagosendines A – C,New Metal Complexes of Imidazole Alkaloids from the Calcareous Sponge Leucetta chagosensis

Chemical examination of the bright yellow sponge Leucetta chagosensis resulted in the isolation of three new imidazole‐based alkaloid complexes namely chagosendines A – C ( 1  –  3 ), together with known analogues pyronaamidine, naamidine J, and naamine C. Their structures were determined on the basis of extensive spectroscopic (IR, MS, NMR, and single‐crystal X‐ray diffraction) analysis in association with the chemical conversion. The isolated alkaloids together with three synthesized new homodimer complexes were evaluated for the cytotoxic activities against a panel of tumor cell lines. The copper complexes of imidazole alkaloids 2 and 3 , as found from nature for the first time, exerted selective and remarkable activities against the tumor cell lines K562, HepG2, and HeLa.     

Polycyclic Polyprenylated Acylphloroglucinols and Chromone O‐Glucosides from Hypericum henryi subsp. uraloides

Two new C(30)‐epimeric polycyclic polyprenylated acylphloroglucinols (PPAPs), named uralodins B and C ( 1 and 2 , resp.), were isolated from the aerial parts of Hypericum henryi subsp. uraloides together with two new chromone glucosides, urachromones A and B ( 3 and 4 , resp.), as well as 16 known compounds. Their structures were established by extensive NMR techniques and MS analysis. The epimers 1 and 2 always behaved like a single compound when examined by TLC, and were separated by HPLC. Their configuration was distinguished by comparative analysis of the NMR data with known analogues together with the ROESY experiment. All the isolated PPAPs were evaluated for their cytotoxic activities against HepG2, SGC7901, HL‐60, and K562 cell lines. Compound 1 showed modest cytotoxic activities against SGC7901 and HL‐60 cell lines, and 2 showed modest cytotoxic activities against HepG2, SGC7901, HL‐60, and K562 cell lines.     

Chaephilones A and B,Two New Azaphilone Derivatives Isolated from Chaetomium globosum

Two new azaphilone derivatives, chaephilones A ( 1 ) and B ( 2 ), were isolated from the fungus Chaetomium globosum, together with four structurally related analogs 3  –  6 . The structures of 1 and 2 were elucidated by comprehensive spectroscopic analyses including HR‐ESI‐MS and NMR. The known compounds were identified as chaetomugilin Q ( 3 ), chaetomugilin D ( 4 ), 11‐epichaetomugilin A ( 5 ), and chaetomugilin S ( 6 ) by comparing their NMR data and optical rotation values with those reported. Compound 2 represents the first example of azaphilone with an open furan ring. Compounds 1 and 2 were evaluated for cytotoxic activities against five human cancer cell lines (HL‐60, SMMC‐7721, A‐549, MCF‐7, and SW480) by the MTS method.     

Diterpenoids from Wedelia prostrata and Their Derivatives and Cytotoxic Activities

One new ent‐kaurane diterpenoid, 11β,16α‐dihydroxy‐ent‐kauran‐19‐oic acid ( 1 ), together with eight known analogues 2 – 9 were isolated from the aerial parts of Wedelia prostrata. One of the acidic diterpenoids, kaurenoic acid ( 3 ), was converted to seven derivatives, 10 – 16 . All compounds were evaluated for their cytotoxic activity in vitro against human leukemia (K562), liver (HepG‐2), and stomach (SGC‐7901) cancer cell lines. Only four kaurenoic acid derivatives, 13 – 16 , with 15‐keto and substitutions at C(19) position, exhibited notable cytotoxic activities on these tumor cell lines with IC₅₀ value ranging from 0.05 to 3.71 μm . Compounds 10 – 12 , with oxime on C(15) showed moderate inhibitory effects and compounds 1 – 9 showed no cytotoxicities on them. Structure–activity relationships were also discussed based on the experimental data obtained. The known derivative, 15‐oxokaurenoic acid 4‐piperdin‐1‐ylbutyl ester ( 17 ), induced typical apoptotic cell death in colon SW480 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis.     

Antimicrobial and Cytotoxic Activities of Lepidium latifolium L. Hydrodistillate,Extract and Its Major Sulfur Volatile Allyl Isothiocyanate

The cultivated Lepidium latifolium L. was investigated to decipher its glucosinolate profile, antimicrobial, and cytotoxic activities. HPLC/ESI‐MS analyses of the intact glucosinolates and GC/MS analysis of their hydrolysis products showed the presence of sinigrin ( 1 ), glucocochlearin ( 2 ), glucotropaeolin ( 3 ), and 4‐methoxyglucobrassicin ( 4 ). Hydrodistillate, extract, and allyl isothiocyanate, the main volatile resulting from sinigrin degradation, showed antimicrobial activity against all eleven tested pathogenic and food spoilage bacteria and fungi, with highest effect observed against Candida albicans with MIC₅₀ 8 and 16 μg/mL. Hydrodistillate and extract showed the best cytotoxic activity on bladder cancer UM‐UC‐3 cell line during an incubation time of 24 h (IC₅₀ 192.9 and 133.8 μg/mL, respectively), while the best effect on glioblastoma LN229 cell line was observed after 48 h (IC₅₀ 110.8 and 30.9 μg/mL, respectively). Pure allyl isothiocyanate displayed a similar trend in cytotoxic effect on both cell lines (IC₅₀ 23.3 and 36.5 μg/mL after 24 h and 48 h, respectively).     

CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE)1

Ammonium is assimilated in algae by the glutamine synthetase (GS)–glutamine:2‐oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1–2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (V_i) was almost completely inhibited in the presence of MSX, suggesting that V_i is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (V_s) rate of ammonium uptake in the presence of 400 μM ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 μM ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 μM ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake.     

Qualitative HPLC‐DAD/ESI‐TOF‐MS Analysis,Cytotoxic, and Apoptotic Effects of Croatian Endemic Centaurea ragusina L. Aqueous Extracts

Centaurea ragusina L., an endemic Croatian plant species, revealed a good cytotoxic activity of aqueous extracts (AE) on human bladder (T24) and human glioblastoma (A1235) cancer cell lines. The chemical constituents were tentatively identified using high performance liquid chromatography HPLC‐DAD/ESI‐TOF‐MS in negative ionization mode. The main compounds of herba extract were sesquiterpene lactones: solstitialin A 3,13‐diacetate and epoxyrepdiolide; organic acid: quinic acid. The main compounds of flower extract were organic acids: quinic acid, citric acid, and malic acid; sesquiterpene lactone: cynaropicrin; phenolic compounds: chlorogenic acid and phenylpropanoid: syringin. The AE of C. ragusina were investigated for correlation of their effects on human bladder (T24) and human glioblastoma (A1235) cancer cell lines using the MTT assay. Although both extracts showed significant dose‐ and time‐dependent cytotoxic activity against both cancer cell lines, the flower extract exhibited slightly higher activity. In order to determine type of cell death induced by treatment, cell lines were exposed subsequently to a treatment with both flower and herba AE. The majority of the cells died by induced apoptosis treatment. Flower AE (26.25%), compared to a leaf AE (22.15%) showed slightly higher percentage of an apoptosis in T24 cells, when compared to a non‐treated cells (0.04%).     

Two New Dolabrane Diterpenes from the Chinese Mangrove Ceriops tagal

Two new dolabrane diterpenes, tagalenes J and K ( 1 and 2 ), together with eleven known analogues ( 3 – 13 ), were isolated from the ethanolic extract of the Chinese mangrove Ceriops tagal. The structures of these compounds were determined by extensive spectroscopic analysis, including 1D‐, 2D‐NMR and HR‐ESI‐MS, as well as the comparison with data in the literatures. Cytotoxicities of isolated compounds against MCF‐7, SW480, HepG2, HeLa, PANC‐1, and A2058 cancer cell lines were also evaluated. Compound 4 exhibited weak cytotoxic activity against SW480, HeLa, and PANC‐1 cell lines with IC₅₀ values of 27.7, 22.2, and 17.6 μm , respectively.     

Four New Diterpene Glucosides from Perovskia atriplicifolia

Four new diterpene glucosides, namely perovskiaditerpenosides A – D ( 1  –  4 ), were isolated from the BuOH extract of Perovskia atriplicifolia. Their structures were well elucidated by chemical methods and comprehensive spectroscopic analyses including MS, IR, and NMR (1D and 2D). The newly isolated compounds were screened for their cytotoxic activity against HepG2, NB4, HeLa, K562, MCF7, PC3, and HL60. The obtained results indicated that the new compounds possessed considerable cytotoxic activity.