Lignin-Derived Syringol and Acetosyringone from Palm Bunch Using Heterogeneous Oxidative Depolymerization over Mixed Metal Oxide Catalysts under Microwave Heating.

Biomass valorization to building block chemicals in food and pharmaceutical industries has tremendously gained attention. To produce monophenolic compounds from palm empty fruit bunch (EFB), EFB was subjected to alkaline hydrothermal extraction using NaOH or K2CO3 as a promotor. Subsequently, EFB-derived lignin was subjected to an oxidative depolymerization using Cu(II) and Fe(III) mixed metal oxides catalyst supported on γ-Al2O3 or SiO2 as the catalyst in the presence of hydrogen peroxide. The highest percentage of total phenolic compounds of 63.87 wt% was obtained from microwave-induced oxidative degradation of K2CO3 extracted lignin catalyzed by Cu-Fe/SiO2 catalyst. Main products from the aforementioned condition included 27.29 wt% of 2,4-di-tert-butylphenol, 19.21 wt% of syringol, 9.36 wt% of acetosyringone, 3.69 wt% of acetovanillone, 2.16 wt% of syringaldehyde, and 2.16 wt% of vanillin. Although the total phenolic compound from Cu-Fe/Al2O3 catalyst was lower (49.52 wt%) compared with that from Cu-Fe/SiO2 catalyst (63.87 wt%), Cu-Fe/Al2O3 catalyst provided the greater selectivity of main two value-added products, syringol and acetosyrigone, at 54.64% and 23.65%, respectively (78.29% total selectivity of two products) from the NaOH extracted lignin. The findings suggested a promising method for syringol and acetosyringone production from the oxidative heterogeneous lignin depolymerization under low power intensity microwave heating within a short reaction time of 30 min.

Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CLpro) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CLpro. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CLpros, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.

Separation and anti-inflammatory evaluation of phytochemical constituents from Pleurospermum candollei (Apiaceae) by high-speed countercurrent chromatography with continuous sample load.

Pleurospermum (Apiaceae) species possess a wide range of biological properties viz. analgesic, anti-inflammatory, antimalarial, and so on. Pleurospermum candollei (DC.) Benth. Ex C. B. Clark. is reported to cure diarrhea, gastric, respiratory, stomach, abdominal, joint, and back pain problems. In addition, it is also used for both male and female infertility. The present study deals with an efficient technique using high-speed countercurrent chromatography for separation of chemical components from the methanol extract of P. candollei. Notably, nine main compounds namely luteolin 7-O-glucoside (1), oxypeucedanin hydrate (2), pabulenol (3), bergapten (4), heptadecanoic acid (5), (E)-isoelemicin (6), trans-asarone (7), α-linolenic acid (8), and isoimperatorin (9) were very efficiently separated and isolated in pure form. Multiple injections were applied followed by two off-line recycling high-speed countercurrent chromatography. The inhibitory effect of nitric oxide production of all compounds was tested in the presence of 200 ng/mL lipopolysaccharide in RAW264.7 mice macrophage cells. The results demonstrated that compounds 7 and 8 effectively inhibited nitric oxide production, with IC50 values of 28.44 and 53.18 µM, respectively. This study thus validates the traditional claim of using P. candollei. Taken together, these findings will be useful in future research to find a potential candidate with anti-inflammatory properties.

Identification of pyrogallol from Awa-tea as an anti-allergic compound that suppresses nasal symptoms and IL-9 gene expression.

As the expression level of allergic disease sensitive genes are correlated with the severity of allergic symptoms, suppression of these gene expressions could be promising therapeutics. We demonstrated that protein kinase Cδ / heat shock protein 90-mediated H1R gene expression signaling and nuclear factor of activated T-cells (NFAT)-mediated IL-9 gene expression signaling are responsible for the pathogenesis of pollinosis. Treatment with Awa-tea combined with wild grape hot water extract suppressed these signaling and alleviated nasal symptoms in toluene-2,4-diisocyanate (TDI)-sensitized rats. However, the underlying mechanism of its anti-allergic activity is not elucidated yet. Here, we sought to identify an anti-allergic compound from Awa-tea and pyrogallol was identified as an active compound. Pyrogallol strongly suppressed ionomycin-induced up-regulation of IL-9 gene expression in RBL-2H3 cells. Treatment with pyrogallol in combination with epinastine alleviated nasal symptoms and suppressed up-regulation of IL-9 gene expression in TDI-sensitized rats. Pyrogallol itself did not inhibit calcineurin phosphatase activity. However, pyrogallol suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFAT. These data suggest pyrogallol is an anti-allergic compound in Awa-tea and it suppressed NFAT-mediated IL-9 gene expression through the inhibition of dephosphorylation of NFAT. This might be the underlying mechanism of the therapeutic effects of combined therapy of pyrogallol with antihistamine. J. Med. Invest. 67 : 289-297, August, 2020.

Simultaneous Determination of Six Compounds in Destructive Distillation Extracts of Hawthorn Seed by GC-MS and Evaluation of Their Antimicrobial Activity.

Hawthorn seed can be used to produce various bioactive compounds through destructive distillation. In this study, an accurate and feasible analytical method based on a gas chromatography mass spectrometer (GC-MS) was developed for simultaneous determination of six major compounds (contributing to more than 3% in total peak area) in destructive distillation extracts of hawthorn seed collected at different temperatures ranging from 150 to 270 °C. Then, a broth microdilution method coupled with grey correlation analysis was engaged in the evaluation of their antimicrobial activities and the screening of primarily active compounds. Results indicate that the extract collected from 211 to 230 °C had the highest content of six major compounds (furfural, 2-methoxyphenol, 2-methoxy-4-methylphenol, 4-ethyl-2-methoxyphenol, 2,6-dimethoxyphenol, and 5-tertbutylpyrogallol) and the strongest antibacterial activity. Besides, 2,6-dimethoxyphenol was found to be a potential compound in inhibiting the growth of vaginitis pathogens. This study provided an optimum temperature for the destructive distillation of hawthorn seed, reducing the waste of energy, and saving the cost of production in the hawthorn industry.

Aromatic compounds produced by endophytic fungi isolated from red alga Asparagopsis taxiformis - Falkenbergia stage.

Endophytic fungi were isolated from red alga Asparagopsis taxiformis - Falkenbergia stage, collected from the Brazilian coast, and were identified as Annulohypoxylon stygium (AT-03) and A. yungensis (AT-06) based on their macro/micromorphological and molecular features. Bioassay-guided fractionation of the EtOAc extract from laboratory cultures of both strains yielded known compounds pyrogallol from A. stygium, (3 R)-scytalone and (3 R,4 R)-4-hydroxy-scytalone from A. yungensis. Pyrogallol was active against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli strains. An inactive fraction from A. stygium afforded two additional compounds, (3 R,4 R)-3,4,5-trihydroxy-1-tetralone and tyrosol. Optically active compounds had their stereochemistry determined by circular dichroism (CD) spectroscopy.

Protective Effect of Pyrogallol-Phloroglucinol-6,6-Bieckol from Ecklonia cava on Monocyte-Associated Vascular Dysfunction.

Ecklonia cava (E. cava) can alleviate vascular dysfunction in diseases associated with poor circulation. E. cava contains various polyphenols with different functions, but few studies have compared the effects of these polyphenols. Here, we comparatively investigated four major compounds present in an ethanoic extract of E. cava. These four major compounds were isolated and their effects were examined on monocyte-associated vascular inflammation and dysfunctions. Pyrogallol-phloroglucinol-6,6-bieckol (PPB) significantly inhibited monocyte migration in vitro by reducing levels of inflammatory macrophage differentiation and of its related molecular factors. In addition, PPB protected against monocyte-associated endothelial cell death by increasing the phosphorylations of PI3K-AKT and AMPK, decreasing caspase levels, and reducing monocyte-associated vascular smooth muscle cell proliferation and migration by decreasing the phosphorylations of ERK and AKT. The results of this study show that four compounds were effective for reduction of monocyte-associated vascular inflammation and dysfunctions, but PPB might be more useful for the treatment of vascular dysfunction in diseases associated with poor circulation.

Induction of HT-29 Colon Cancer Cells Apoptosis by Pyrogallol with Growth Inhibiting Efficacy Against Drug-Resistant Helicobacter pylori.

BACKGROUND: Colon cancer is the most aggressive form of cancers, that causes 0.5 million deaths per year around the globe. Targeting colon cancer by conventional therapeutic options elicits toxicity. Traditional medicines take a lead to alleviate the existing clinical challenges. OBJECTIVE: To investigate antibacterial activity against Helicobacter Pylori and in vitro anti-colon cancer activity by Acacia nilotica extract (ACE) and its active constituent pyrogallol. METHODS: Pyrogallol isolated from A. nilotica by column chromatography and HPLC and structure was elucidated by spectral analysis. Antibacterial activity was done by flow cytometry. Cytotoxicity was measured by MTT assay. Apoptotic morphology and nuclear fragmentation were assessed with AO/ethidium bromide and DAPI staining. DNA fragmentation was done by electrophoresis. Western blot used to analyze the molecular mechanism of apoptosis. Cell cycle arrest was determined using flow cytometry of propidium iodide stained cells. Cell migration was determined by wound healing assay. RESULTS: ACE (20 µg/ml) and pyrogallol (10 µg/ml) treatment reduced the survival of H.pylori at 61% and 62%, respectively. MTT results show that HT-29 cells are more sensitive to pyrogallol with an IC50 value of 35µg/ml compared to ACE. Pyrogallol treated HT-29 cells reached dead state i.e. late apoptotic state with severe nuclear fragmentation. Pyrogallol elicits dose dependent DNA fragmentation in HT-29 cells. Pyrogallol induced apoptosis by simultaneous down-regulation of Bcl-2 and up-regulation of BAX and cytochrome c. Pyrogallol arrested HT-29 cells in S and G2/M phase of cell cycle. Further pyrogallol exhibited marked antimetastatic potential by inhibiting the migration of HT-29 cells dose dependently. CONCLUSION: Both ACE and pyrogallol repressed the growth of H.pylori and as significant anti-colon cancer agent.

Insecticidal Activity of the Leaf Essential Oil of Peperomia borbonensis Miq. (Piperaceae) and Its Major Components against the Melon Fly Bactrocera cucurbitae (Diptera: Tephritidae).

The essential oil from the leaves of Peperomia borbonensis from Réunion Island was obtained by hydrodistillation and characterized using GC-FID, GC/MS and NMR. The main components were myristicin (39.5%) and elemicin (26.6%). The essential oil (EO) of Peperomia borbonensis and its major compounds (myristicin and elemicin), pure or in a mixture, were evaluated for their insecticidal activity against Bactrocera cucurbitae (Diptera: Tephritidae) using a filter paper impregnated bioassay. The concentrations necessary to kill 50% (LC50 ) and 90% (LC90 ) of the flies in three hours were determined. The LC50 value was 0.23 ± 0.009 mg/cm2 and the LC90 value was 0.34 ± 0.015 mg/cm2 for the EO. The median lethal time (LT50 ) was determined to compare the toxicity of EO and the major constituents. The EO was the most potent insecticide (LT50  = 98 ± 2 min), followed by the mixture of myristicin and elemicin (1.4:1) (LT50  = 127 ± 2 min) indicating that the efficiency of the EO is potentiated by minor compounds and emphasizing one of the major assets of EOs against pure molecules.

Pyrogallol abates VSMC migration via modulation of Caveolin-1, matrix metalloproteinase and intima hyperplasia in carotid ligation mouse.

Migration of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia and other vascular diseases. Caveolin-1 (Cav-1) has been recognized as a proliferative inhibitor of VSMCs and is likely to be an important regulator of VSMC migration. The underlying mechanism of pyrogallol on the VSMC migration is not fully understood. This study attempted to dissect the role of Cav-1 and matrix metalloproteinase (MMP) in VSMC migration and to investigate the effect of pyrogallol on VSMC mobility during carotid artery ligation mice. The mRNA expression of MMP-3 and MMP-13 was down-regulated in cultured VSMC prepared from Cav-1-deficient (Cav-1 KO) mice whereas MMP-14 expression was up-regulated. Pyrogallol effectively inhibited the migration of Cav-1 KO VSMC by promoting the expression of tissue inhibitors of metalloproteinase (TIMP)-2. Pyrogallol also inhibited the migration of Cav-1 wild type (WT) VSMC, however, by increasing TIMP-1 expression and repressing MMP-2 activity. In a parallel in vivo study, intra-peritoneal (ip) of pyrogallol to carotid artery ligated mice significantly suppressed intima formation in mice carotid artery. Furthermore, the proMMP-9 activity in pyrogallol-treated mice serum significantly increased from Day 0 to Day 2 and decreased from Day 2 to Day 7 in a time-dependent manner. In addition, WT mice treated with pyrogallol had significantly reduced neointima formation, whereas no differences were observed in Cav-1 knock out (KO) mice. These results suggest that pyrogallol not only inhibited VSMC migration but also effectively diminishes the severity of neointima hyperplasia, implying that pyrogallol possesses potential anti-atherogenic effects for the treatment of vascular diseases.

Separation of high-purity syringol and acetosyringone from rice straw-derived bio-oil by combining the basification-acidification process and column chromatography.

Numerous technologies have been used to reclaim valuable chemicals from bio-oil. In this study, a combination of the basification-acidification process and column chromatography was employed for the separation of high-purity syringol and acetosyringone from rice straw-derived bio-oil. The optimal conditions for the basification-acidification process and the possible precipitation mechanism of the basification were explored. The results showed the following as the optimal conditions for the basification process: mass ratio of calcium hydroxide (Ca(OH)2 ) to bio-oil, 2.0; reaction temperature, 70°C; and reaction time, 30 min. The results also showed that 1.6 mol of hydrochloric acid (HCl) per gram of bio-oil was optimal for the acidification. The precipitation was found to proceed via a possible mechanism involving the reaction of the phenolic compounds in the bio-oil with Ca(OH)2 to produce a precipitate. After further separation by column chromatography, purities of 91.4 and 96.2% (from gas chromatography-mass spectrometry) were obtained for syringol and acetosyringone, respectively. Their recoveries for the whole process were 73.0 and 39.3%, respectively.

HPLC-Guided Isolation, Purification and Characterization of Phenylpropanoid and Phenolic Constituents of Nutmeg Kernel (Myristica fragrans).

Many studies on the biological activities of nutmeg continue to appear in the literature. The most common targets include GIT, CNS, oxidative stress and inflammation. However, results obtained from most studies are often inconsistent due to the variability of utilized samples, lack of standardized nutmeg products or insufficient amounts of pure compounds for comprehensive follow-up investigation. To address the consistency and supply issue we utilized available technology to develop a reproducible procedure for preparation of specific extracts and isolation of the major phenolic constituents present in nutmeg kemel. A well-defined and reproducible sequence of extraction, fractionation and chromatographic purification was adopted and was guided by HPLC fingerprinting. Spectroscopic methods, mainly NMR, and mass spectrometry were utilized to identify each compound. Thirteen compounds were isolated in a pure form and identified as: elemicin (1), isoelemicin (2), myristicin (4), surinamensin (5), malabaricone C (6), 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l- acetoxy-(3,4-dimethoxyphenyl)-propyl ester (7), methoxylicarin A (8), licarin A (9), malabaricone B (10), licarin C (11), 5'-methoxylicarin B (12), licarin B (13), and 2-(3'-allyl-2',6'-dimethoxy-phenyloxy)-l-methyl-5-methoxy-1,2-dihydrobenzofuran (3, a new compound). With repeated isolation runs, these pure compounds can be prepared in quantities sufficient for biological evaluation as needed. The availability of purified compounds will also allow the development of specific, accurate, and sensitive analytical procedures for pharmacokinetic studies and for quality control of nutmeg products. Both aspects are essential for nutmeg-focused drug discovery. The same approach can also be adapted to other medicinal plants of potential interest.

Isolation and identification of oxidation products of syringol from brines and heated meat matrix.

In this study we developed new extraction and detection methods (using HPLC-UV and LC-MS), making it possible to analyze the smoke phenol syringol and its oxidation products nitrososyringol, nitrosyringol, and the syringol dimer 3,3',5,5'-tetramethoxy-1,1'-biphenyl-4,4'-diol, which were identified in heated meat for the first time. Preliminary brine experiments performed with different concentrations of ascorbic acid showed that high amounts of this antioxidant also resulted in almost complete degradation of syringol and to formation of the oxidation products when the brines were heated at low pH values. Heat treatment (80°C) and subsequent simulated digestion applied to meat samples containing syringol, ascorbic acid and different concentrations of sodium nitrite produced 3,3',5,5'-tetramethoxy-1,1'-biphenyl-4,4'-diol even at a low nitrite level in the meat matrix, while nitroso- and nitrosyringol were isolated only after the digestion experiments. Increasing amounts of oxygen in the meat matrix decreased the syringol concentration and enhanced the formation of the reaction products in comparison to the samples without added oxygen.

Chemical composition, antioxidant, antitumor, anticancer and cytotoxic effects of Psidium guajava leaf extracts.

Context Psidium guajava L. (Myrtaceae) leaves are used in traditional medicines for the treatment of cancer, inflammation and other ailments. Objective The current study explores scientific validation for this traditional medication. Materials and methods We used ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryl hydrazil (DPPH) assays to estimate antioxidant activity of P. guajava leaf extracts (methanol, hexane and chloroform). Antitumour and in vivo cytotoxic activities were determined using potato disc assay (PDA) and brine shrimp lethality assay, respectively. Three human carcinoma cell lines (KBM5, SCC4 and U266) were incubated with different doses (10-100 µg/mL) of extracts and the anticancer activity was estimated by MTT assay. NF-κB suppressing activity was determined using electrophoretic mobility shift assay (EMSA). Chemical composition of the three extracts was identified by GC-MS. Total phenolic and flavonoid contents were measured by colorimetric assays. Results and discussions The order of antioxidant activity of three extracts was methanol > chloroform > hexane. The IC50 values ranged from 22.73 to 51.65 µg/mL for KBM5; 22.82 to 70.25 µg/mL for SCC4 and 20.97 to 89.55 µg/mL for U266 cells. The hexane extract exhibited potent antitumour (IC50 value = 65.02 µg/mL) and cytotoxic (LC50 value = 32.18 µg/mL) activities. This extract also completely inhibited the TNF-α induced NF-κB activation in KBM5 cells. GC-MS results showed that pyrogallol, palmitic acid and vitamin E were the major components of methanol, chloroform and hexane extracts. We observed significant (p < 0.05) difference in total phenolic and flavonoid contents of different solvent extracts. Conclusion The present study demonstrates that P. guajava leaf extracts play a substantial role against cancer and down-modulate inflammatory nuclear factor kB.

Three new sulphur glycosides from the seeds of Descurainia sophia.

Three new sulphur glycosides, raphanuside B-D (1-3), together with a known sulphur glycoside, raphanuside (4) were isolated from the decoction of the seeds of Descurainia sophia (L.) Webb ex Prantl, and the compound 4 was reported for the first time from this plant. Their structures were identified by means of UV, IR, 1D, 2D NMR (HSQC, HMBC and NOESY) and HR-ESI-MS spectroscopic data.

GC-MS method for determination and pharmacokinetic study of four phenylpropanoids in rat plasma after oral administration of the essential oil of Acorus tatarinowii Schott rhizomes.

ETHNOPHARMACOLOGICAL RELEVANCE: Acorus tatarinowii Schott (AT), belong to the family Araceae, is perennial herbaceous plant mainly present in China, Japan and India. The rhizomes of AT have been used as a famous traditional Chinese medicine for the treatment of central nervous system related diseases. AIM OF THE STUDY: A selective, accurate and sensitive method using gas chromatography-mass spectroscopy (GC-MS) for the simultaneous determination and pharmacokinetic study of ß-asarone, α-asarone, elemicin and cis-methyl isoeugenol in rat plasma was developed and validated. MATERIALS AND METHODS: The GC-MS system was operated under selected ion monitoring (SIM) mode. The samples were prepared by protein precipitation with acetonitrile after being spiked with an internal standard (1-naphthol). The GC separation was achieved on a DB-1701 column (60 m × 0.25 mm ID, and 0.25 µm film thickness). RESULTS: The current GC/MS assay was validated for linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability. The analyte calibration curves were linear over a wide concentration range and the lowest limit of quantifications (LLOQ) were 5.53 ng/mL (ß-asarone), 6.50 ng/mL (α-asarone), 3.10 ng/mL (elemicin) and 7.60 ng/mL (cis-methyl isoeugenol). After oral administration 0.9 g /Kg of AT rhizomes, the maximum plasma concentration (Cmax) was 2508.6±498.7 ng/mL for ß-asarone, 257.5±37.1 ng/mL for α -asarone, 345.5±33.4 ng/mL for elemicin and 452.7±59.1 ng/mL for cis-methyl isoeugenol, respectively. The time to reach the maximum plasma concentration (Tmax) was 1.42±0.18 h for ß-asarone, 1.58±0.19 h for α -asarone, 1.67±0.24 h for elemicin and 1.75±0.38 h for cis-methyl isoeugenol, respectively. CONCLUSION: This paper described a simple, sensitive and validated GC-MS method for simultaneous determination of four phenylpropanoids in rat plasma after oral administration of the essential oil of AT rhizomes and investigated on their pharmacokinetics studies as well.

Nakijiquinone S and nakijinol C, new meroterpenoids from a marine sponge of the family spongiidae.

New meroterpenoids, nakijiquinone S (1) and nakijinol C (2), have been isolated from an Okinawan marine sponge of the family Spongiidae. The gross structures and relative stereochemistries of 1 and 2 were elucidated on the basis of their spectral data. Nakijiquinone S (1) and nakijinol C (2) were new meroterpenoids consisting of a clerodane-type decalin ring connected to a 2-butoxy-5-hydroxy-benzoquinone unit or methyl 2,3,4-trihydroxybenzoate unit through a methylene, respectively. Nakijiquinone S (1) and nakijinol C (2) showed antimicrobial activities against several bacteria and fungi.

GC/GC-MS analysis, isolation and identification of bioactive essential oil components from the Bhutanese medicinal plant, Pleurospermum amabile.

We have hydrodistilled the essential oil (EO) from the aerial parts of the Bhutanese medicinal plant, Pleurospermum amabile using a Clevenger apparatus and evaluated this EO by GC/GC-MS and NMR analysis followed by testing for bioactivity. The GC-MS analysis identified 52 compounds with (E)-isomyristicin as a major component (32.2%). Repeated purification yielded four compounds; (E)-isomyristicin (1), (E)-isoapiol (2), methyl eugenol (3) and (E)-isoelemicin (4). Compound 2 and the mother EO showed the best antiplasmodial activity against the Plasmodium falciparum strains, TM4/8.2 (chloroquine and antifolate sensitive) and K1CB1 (multidrug resistant). They exhibited mild antibacterial activity against Bacillus subtilis. None of the test samples showed cytotoxicity.

Ultraviolet spectroscopy of fundamental lignin subunits: guaiacol, 4-methylguaiacol, syringol, and 4-methylsyringol.

Ultraviolet spectroscopy of the G- and S-type lignin subunits, guaiacol (G) and syringol (S), along with their para-methylated derivatives 4-methylguaiacol (4-MG) and 4-methylsyringol (4-MS), has been carried out in the cold, isolated environment of a supersonic jet. The excitation spectra and dispersed fluorescence (DFL) spectra of G and 4-MG show strong S0-S1 origins and Franck-Condon activity involving both the ring modes typical of aromatic derivatives, and the four lowest frequency out-of-plane modes (a") and lowest in-plane mode (a') involving the OH and OCH3 groups. The four low-frequency out-of-plane modes undergo extensive Duschinsky mixing between the ground and excited state. In 4-MG, combination bands involving methyl rotor levels with out-of-plane modes appeared with surprisingly high intensity, indicating a high degree of hindered rotor-vibration coupling in both S0 and S1. These mixing effects accompany the change in geometry upon π-π∗ electronic excitation going from a planar ground state to a non-planar excited state. Time-dependent density functional theory (TDDFT M05-2X∕6-311++G(d,p)) calculations predict a geometric distortion along the out-of-plane oxygen flapping coordinate, yielding a double minimum potential in S1 with a barrier to planarity of 195 cm(-1) in G. The excitation spectrum of S and 4-MS showed a much higher degree of spectral congestion and a larger geometry change evident by a shifted intensity distribution peaking ∼300 cm(-1) above the electronic origin. TDDFT calculations predict a larger geometry change in S compared with G, with the OH and H-bonded methoxy groups displaced in opposite directions above∕below the ring plane. Dispersed fluorescence from all S1 excited state levels in S∕4-MS yield only broad emission peaking far to the red of the excitation wavelength (-4500 cm(-1)). Several hypotheses regarding the source of this broad, redshifted emission were tested, but the cause remains unclear. p-Methylation was found to significantly redshift the UV absorption in both 4-MG and 4-MS, and methyl rotor transitions were assigned in both allowing for the determination of the shape and barrier heights of their respective potentials. These results provide a foundation for the discrimination of G- and S-chromophores in lignin oligomers, and demonstrate the potential for site-selective absorption.

Generation of reactive oxygen species in cyanobacteria and green algae induced by allelochemicals of submerged macrophytes.

Inhibition of phytoplankton by allelochemicals released by submerged macrophytes is reported to be one of the mechanisms that maintain a clear-water state in shallow lakes. In order to elucidate this mechanism, the ability of six polyphenols and two long-chain fatty acids to induce the generation of reactive oxygen species (ROS) in phytoplankton was studied using the ROS sensitive probe 2',7'- dichlorodihydrofluorescein diacetate (DCFH-DA). The results showed that only (+)-catechin (CA) and pyrogallic acid (PA) could induce ROS formation in Microcystis aeruginosa and Pseudokirchneriella subcapitata. 25 mg L⁻¹ CA caused 1.2, 1.4 and 1.8 times increase of ROS levels in M. aeruginosa at 1, 2 and 4h exposure, respectively, and, correspondingly in P. subcapitata cells, these values were 3.7, 6.2 and 7.7, respectively. PA also significantly increased the levels of intracellular ROS in P. subcapitata (P < 0.01); however, significant ROS generation in M. aeruginosa was observed at only 4h exposure (P < 0.01). Light enhanced ROS generation in CA treated cells, but not in the cells treated with PA. CA and PA may act as redox cyclers after uptake by test organisms and produce ROS successively. These results suggest that the oxidative stress induced by the redox cycling property of allelochemicals may be one of the important causes for the inhibitory effect of some submerged macrophytes towards undesired phytoplankton in natural aquatic ecosystems.