Coniferyl aldehyde alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via JAK2-STAT1 pathway in acute pneumonia.

Pneumonia is a kind of inflammatory disease characterized by pathogen infection of lower respiratory track. Lipopolysaccharide (LPS) is the main bioactive component of Gram-negative bacteria responsible for inflammatory response. Recently, coniferyl aldehyde (CA) has been reported to play a crucial role because of its anti-inflammatory activity. However, the effect and mechanisms of CA in ameliorating symptoms of acute pneumonia remain unknown. Evaluating and identifying the value and exploring the mechanisms of CA on LPS-mediated WI-38 apoptosis and inflammation were the aims of this study. Here, CCK-8 cell viability assay was applied on WI-38 after treatment with or without LPS at different doses of CA to verify that CA can increase LPS-induced cell viability. Then, quantitative polymerase chain reaction (qPCR) and enzyme-linked-immunosorbent serologic assays (ELISA) suggested that LPS treatment dramatically decreased the expression level of IL-10 (anti-inflammatory factor) while strikingly increasing the expression levels of IL-1ß, IL-6, and TNF-α (tumor necrosis factor-α; proinflammatory factor) whereas CA treatment attenuates LPS-induced inflammation of WI-38. Further, flow cytometry and Western blot assay verified that LPS treatment dramatically promoted apoptosis of WI-38 cells, while administration of CA notably inhibited apoptosis of WI-38 cells. Moreover, the Western blot assay hinted that CA could inactivate LPS-induced JAK2-STAT1 signaling pathway. These findings indicated that CA could alleviate LPS-mediated WI-38 apoptosis and inflammation injury through JAK2-STAT1 pathway in acute pneumonia.

Coniferyl aldehyde alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via JAK2–STAT1 pathway in acute pneumonia

Pneumonia is a kind of inflammatory disease characterized by pathogen infection of lower respiratory track. Lipopolysaccharide (LPS) is the main bioactive component of Gram-negative bacteria responsible for inflammatory response. Recently, coniferyl aldehyde (CA) has been reported to play a crucial role because of its anti-inflammatory activity. However, the effect and mechanisms of CA in ameliorating symptoms of acute pneumonia remain unknown. Evaluating and identifying the value and exploring the mechanisms of CA on LPS-mediated WI-38 apoptosis and inflammation were the aims of this study. Here, CCK-8 cell viability assay was applied on WI-38 after treatment with or without LPS at different doses of CA to verify that CA can increase LPS-induced cell viability. Then, quantitative polymerase chain reaction (qPCR) and enzyme-linked-immunosorbent serologic assays (ELISA) suggested that LPS treatment dramatically decreased the expression level of IL-10 (anti-inflammatory factor) while strikingly increasing the expression levels of IL-1β, IL-6, and TNF-α (tumor necrosis factor-α; proinflammatory factor) whereas CA treatment attenuates LPS-induced inflammation of WI-38. Further, flow cytometry and Western blot assay verified that LPS treatment dramatically promoted apoptosis of WI-38 cells, while administration of CA notably inhibited apoptosis of WI-38 cells. Moreover, the Western blot assay hinted that CA could inactivate LPS-induced JAK2–STAT1 signaling pathway. These findings indicated that CA could alleviate LPS-mediated WI-38 apoptosis and inflammation injury through JAK2–STAT1 pathway in acute pneumonia (AU)

Tolerance of Yarrowia lipolytica to inhibitors commonly found in lignocellulosic hydrolysates.

BACKGROUND: Lignocellulosic material is a suitable renewable carbon and energy source for microbial cell factories, such as Yarrowia lipolytica. To be accessible for microorganisms, the constituent sugars need to be released in a hydrolysis step, which as a side effect leads to the formation of various inhibitory compounds. However, the effects of these inhibitory compounds on the growth of Y. lipolytica have not been thoroughly investigated. RESULTS: Here we show the individual and combined effect of six inhibitors from three major inhibitor groups on the growth of Y. lipolytica. We engineered a xylose consuming strain by overexpressing the three native genes XR, XDH, and XK and found that the inhibitor tolerance of Y. lipolytica is similar in glucose and in xylose. Aromatic compounds could be tolerated at high concentrations, while furfural linearly increased the lag phase of the cultivation, and hydroxymethylfurfural only inhibited growth partially. The furfural induced increase in lag phase can be overcome by an increased volume of inoculum. Formic acid only affected growth at concentrations above 25 mM. In a synthetic hydrolysate, formic acid, furfural, and coniferyl aldehyde were identified as the major growth inhibitors. CONCLUSION: We showed the individual and combined effect of inhibitors found in hydrolysate on the growth of Y. lipolytica. Our study improves understanding of the growth limiting inhibitors found in hydrolysate and enables a more targeted engineering approach to increase the inhibitor tolerance of Y. lipolytica. This will help to improve the usage of Y. lipolytica as a sustainable microbial cell factory.

A rapid and simple ultra high performance liquid chromatography method for the simultaneous determination of methoxyphenol derivatives involved in the eugenol catabolic pathway.

An efficient ultra high performance liquid chromatography method of separation was developed for the analysis of six important methoxyphenol derivatives involved in the eugenol catabolic pathway. In the present study, an Acquity UPLC BEH C18 column was used for the chromatographic separation of the industrially important phenolic compounds such as vanillin, vanillic acid, ferulic acid, coniferyl alcohol, and coniferyl aldehyde obtained during microbial transformation of eugenol. Eluted components were identified using the dual wavelength (254 and 310 nm) UV detector. A gradient method of elution using mobile phase of aqueous 1 mM trifluoroacetic acid (Solvent A) and methanol (Solvent B) at a flow rate of 0.3 mL/min separated all the five intermediate methoxyphenol derivatives along with their precursor eugenol within 15 min with stable baseline resolution. Method validation was performed for the accurate quantification of vanillin, coniferyl aldehyde, and eugenol using the parameters of linearity, specificity, precision, limit of detection, limit of quantification, and robustness. The developed method would be helpful for clear separation and identification of the five most important intermediate metabolites of the eugenol catabolism pathway.

Infrared and Raman spectra of lignin substructures: Coniferyl alcohol, abietin, and coniferyl aldehyde.

Anatomical and chemical information can be linked by Raman imaging. Behind every pixel of the image is a Raman spectrum, which contains all the information as a molecular fingerprint. Yet to understand the spectra, the bands have to be assigned to components and their molecular structures. Although the lignin distribution is easily tracked in plant tissues, the assignment of the spectra is not good enough to allow in-depth analysis of the composition. Assignments of three lignin model compounds were derived from polarization measurements and quantum-chemical computations. Raman spectra of coniferyl alcohol crystals showed orientation dependence, which helped in band assignment. Abietin showed a Raman spectrum that was very similar to the spectrum of coniferyl alcohol, whereas its IR spectrum was very different due to bands of the sugar moiety. The Raman spectrum of coniferyl aldehyde is affected by the crystal order of molecules. All three compounds show much stronger band intensities than unconjugated single aromatic rings, indicating that the bulk of the lignin structure has significantly reduced contribution to Raman band intensities. Therefore, it is possible to highlight certain structures of lignin with Raman spectroscopy, because low amounts of a compound do not necessarily mean weak features in the spectrum.

Genomic and transcriptomic analysis of a coniferyl aldehyde-resistant Saccharomyces cerevisiae strain obtained by evolutionary engineering.

Phenolic inhibitors in lignocellulosic hydrolysates interfere with the performance of fermenting microorganisms. Among these, coniferyl aldehyde is one of the most toxic inhibitors. In this study, genetically stable Saccharomyces cerevisiae mutants with high coniferyl aldehyde resistance were successfully obtained for the first time by using an evolutionary engineering strategy, based on the systematic application of increasing coniferyl aldehyde stress in batch cultures. Among the selected coniferyl aldehyde-resistant mutants, the highly resistant strain called BH13 was also cross-resistant to other phenolic inhibitors, vanillin, ferulic acid and 4-hydroxybenzaldehyde. In the presence of 1.2 mM coniferyl aldehyde stress, BH13 had a significantly reduced lag phase, which was less than 3 h and only about 25% of that of the reference strain and converted coniferyl aldehyde faster. Additionally, there was no reduction in its growth rate, either. Comparative transcriptomic analysis of a highly coniferyl aldehyde-resistant mutant revealed upregulation of the genes involved in energy pathways, response to oxidative stress and oxidoreductase activity in the mutant strain BH13, already under non-stress conditions. Transcripts associated with pleiotropic drug resistance were also identified as upregulated. Genome re-sequencing data generally supported transcriptomic results and identified gene targets that may have a potential role in coniferyl aldehyde resistance.

Direct biodegradation of eugenol to coniferyl aldehyde and other higher value-added products by Gibberella fujikuroi ZH-34

BACKGROUND: Eugenol is an economically favorable substrate for the microbial biotransformation of aromatic compounds. Coniferyl aldehyde is one kind of aromatic compound that is widely used in condiment and medical industries; it is also an important raw material for producing other valuable products such as vanillin and protocatechuic acid. However, in most eugenol biotransformation processes, only a trace amount of coniferyl aldehyde is detected, thus making these processes economically unattractive. As a result, an investigation of new strains with the capability of producing more coniferyl aldehyde from eugenol is required. RESULTS: We screened a novel strain of Gibberella fujikuroi, labeled as ZH-34, which was capable of transforming eugenol to coniferyl aldehyde. The metabolic pathway was analyzed by high-performance liquid chromatography­mass spectrometry and transformation kinetics. The culture medium and biotransformation conditions were optimized. At a 6 h time interval of eugenol fed-batch strategy, 3.76 ± 0.22 g/L coniferyl aldehyde was obtained, with the corresponding yield of 57.3%. CONCLUSIONS: This work improves the yield of coniferyl aldehyde with a biotechnological approach. Moreover, the fed-batch strategy offers possibility for controlling the target product and accumulating different metabolites

Chemical constituents from stems of Acanthopanax henryi / 中草药

Objective To study the chemical constituents from stems of Acanthopanax henryi based on LPS-induced macrophages RAW264.7 and microglia BV2 as the bioactivity guided model. Methods The compounds were isolated and purified by silica gel and Sephadex LH-20 column chromatography, as well as Prep-TLC and recrystallization methods. Their structures were identified on the basis of their physicochemical properties and spectroscopic data. Results Eighteen compounds were obtained from A. henryi and their chemical structures were identified as p-hydroxybenzoic acid (1), trans-p-hydroxycinnamic acid (2), (E)-caffeic acid methyl ester (3), caffeic acid (4), trans-coniferyl aldehyde (5), syringaldehyde (6), vanillin (7), 6-methoxy-7-hydroxycoumarin (8), trans-sinapaldehyde (9), undecane-1,11-dioic acid monomethyl ester (10), (-)-sesamin (11), 3-O-caffeoyl-quinic acid (12), 5-O-caffeoyl-quinic acid (13), 1,3-di-O-caffeoyl-quinic acid (14), 1,4-di-O-caffeoyl-quinic acid (15), 1,5-di-O-caffeoyl-quinic acid (16), stigmasterol (17), and β-sitosterol (18), respectively. Conclusion To the best of our knowledge, compound 10 was isolated from Araliaceae for the first time. Except compounds 12, 14, 17, and 18, all of other compounds were obtained from this species for the first time.

Chemical constituents of Phyllanthus emblica and its anti-inflammation activities / 中草药

Objective: To study the chemical constituents and its anti-inflammtory activity effect of Phyllanthus emblica. Methods: The chemical constituents of P. emblica were isolated and purified by silica gel column chromatography, ODS column chromatography, Sephadex LH-20 column chromatography, semi-preparative high-performance liquid chromatography and recrystallization method. Through their spectra data, physical and chemical properties analysis, the structures of those compounds with high content were identified. LPS-induced RAW264.7 inflammatory cell model was established to evaluate the effect of compounds in P. emblica on proinflammatory factors (NO, IL-6, TNF-α, and MCP-1) of RAW264.7 inflammatory cells. Results: Totally, 14 compounds were isolated from P. emblica. and idetified as isovanillic acid (1), trans-cinnamic acid (2), p-hydroxybenzaldehyde (3), coniferyl aldehyde (4), quercetin (5), kaempferol-3-O-α-L-rhamnose (6), naringenin (7), 2-hydroxy-3-methyl phenylpropiolate (8), hydroquinone (9), myricetin (10), 2-furoic acid (11), methyl gallate (12), protocatechuic acid (13), gallic acid (14). The experiment of anti-inflammatroy effects showed that those compounds had different inhibitory effects on the production of inflammatory factors NO, IL-6, TNF-α and MCP-1. Conclusion: Compounds 1, 3, 4, 8-11 and 13 are isolated from P emblica for the first time. The anti-inflammatory effect of P. emblica is related to its phenolic acids.

Stereoisomeric guaiacylglycerol-ß-coniferyl aldehyde ether induces distinctive apoptosis by downregulation of MEK/ERK pathway in hepatocellular carcinoma cells.

Two 8-O-4'-type neolignan epimers erythro-guaiacylglycerol-ß-coniferyl aldehyde ether (1) and threo-guaiacylglycerol-ß-coniferyl aldehyde ether (2) were isolated from the stems of Picrasma quassioides. Further chiral separation gave two pairs of enantiomers 1a/1b and 2a/2b. The cytotoxicity assay against hepatocellular carcinoma Hep3B and HepG2 cells was evaluated by MTT assay. The results showed that 1b (IC50 = 45.56 µM) and 2b (IC50 = 39.02 µM) had more cytotoxic effect than its enantiomers 1a (IC50 = 82.66 µM) and 2a (IC50 = 67.97 µM) in Hep3B cells, respectively. Moreover, 1b and 2b could induce more apoptotic cells as well as higher reactive oxygen species (ROS) generation than 1a and 2a at 50 µM. In addition, a further study on the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways was investigated. The results revealed that all compounds had no significant effect on PI3K/AKT pathway, however, 1b and 2b attenuated the relative levels of p-MEK and p-ERK when compared with 1a and 2a. Taken together, the absolute configurations of guaiacylglycerol-ß-coniferyl aldehyde ether had an impact on the inhibitory effect on Hep3B cells. The inactivation of MEK/ERK signaling pathway might contribute to apoptosis induction and ROS generation in 1b- and 2b-treated cells.

Selective inhibition of JAK2/STAT1 signaling and iNOS expression mediates the anti-inflammatory effects of coniferyl aldehyde.

Urgent needs still exist for selective control of excessive inflammation. Despite the therapeutic potential of natural compounds against inflammation-associated chronic conditions, lack of specific molecular targets renders these bioactive compounds difficult for further development. Here we examined the bioactivity of coniferyl aldehyde (CA), a natural phenolic compound found in several dietary substances and medicinal plants, elucidating its efficacy both in vivo and in vitro with underlying molecular mechanisms. IFN-γ/TNF-α-stimulated human keratinocytes and lipopolysaccharide (LPS)-stimulated murine macrophages were used to examine the effect of CA in vitro and to elucidate the underlying mechanisms. In vivo models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and carrageenan (CRG)-induced paw edema were employed to investigate the topical and systemic anti-inflammatory effects of CA, respectively. CA significantly reduced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated macrophages. While nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) pathways, the representative cellular pathways for iNOS induction, were not affected by CA, phosphorylation of Janus kinase 2 (JAK2) and signal Transducers and Activators of Transcription 1 (STAT1) and subsequent nuclear translocation of p-STAT1 were significantly decreased by CA. The effect of CA on JAK2-STAT1-iNOS axis was also observed in human keratinocytes stimulated with IFN-γ/TNF-α. Topical application of CA to mice produced significant protection against TPA-induced ear edema along with suppressed epidermal hyperproliferation and leucocyte infiltration. Systemic administration of CA significantly reduced CRG-induced paw edema in rats, where CRG-induced iNOS expression and STAT1 phosphorylation were decreased by CA. In summary, CA has significant anti-inflammatory properties both in vitro and in vivo, mediated by significant selective inhibition of JAK2-STAT1-iNOS signaling. CA is an attractive novel candidate for treating inflammatory diseases associated with excessive production of NO.

A coniferyl aldehyde dehydrogenase gene from Pseudomonas sp. strain HR199 enhances the conversion of coniferyl aldehyde by Saccharomyces cerevisiae.

The conversion of coniferyl aldehyde to cinnamic acids by Saccharomyces cerevisiae under aerobic growth conditions was previously observed. Bacteria such as Pseudomonas have been shown to harbor specialized enzymes for converting coniferyl aldehyde but no comparable enzymes have been identified in S. cerevisiae. CALDH from Pseudomonas was expressed in S. cerevisiae. An acetaldehyde dehydrogenase (Ald5) was also hypothesized to be actively involved in the conversion of coniferyl aldehyde under aerobic growth conditions in S. cerevisiae. In a second S. cerevisiae strain, the acetaldehyde dehydrogenase (ALD5) was deleted. A prototrophic control strain was also engineered. The engineered S. cerevisiae strains were cultivated in the presence of 1.1mM coniferyl aldehyde under aerobic condition in bioreactors. The results confirmed that expression of CALDH increased endogenous conversion of coniferyl aldehyde in S. cerevisiae and ALD5 is actively involved with the conversion of coniferyl aldehyde in S. cerevisiae.

Lignans and aromatic glycosides from Piper wallichii and their antithrombotic activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper wallichii (Miq.) Hand.-Mazz. is a medicinal plant used widely for the treatment of rheumatoid arthritis, inflammatory diseases, cerebral infarction and angina in China. Previous study showed that lignans and neolignans from Piper spp. had potential inhibitory activities on platelet aggregation. In the present study, we investigated the chemical constituents of Piper wallichii and their antithrombotic activities, to support its traditional uses. MATERIALS AND METHODS: The methanolic extract of the air-dried stems of Piper wallichii was separated and purified using various chromatographic methods, including semi-preparative HPLC. The chemical structures of the isolates were determined by detailed spectroscopic analysis, and acidic hydrolysis in case of the new glycoside 2. Determination of absolute configurations of the new compound 1 was facilitated by calculated electronic circular dichroism using time-dependent density-functional theory. All compounds were tested for their inhibitory effects on platelet aggregation induced by platelet activating factor (PAF) in rabbits׳ blood model, from which the active ones were further evaluated the in vivo antithrombotic activity in zebrafish model. RESULTS: A new neolignan, piperwalliol A (1), and four new aromatic glycosides, piperwalliosides A-D (2-5) were isolated from the stems of Piper wallichii, along with 25 known compounds, including 13 lignans, six aromatic glycosides, two phenylpropyl aldehydes, and four biphenyls. Five known compounds (6-10) showed in vitro antiplatelet aggregation activities. Among them, (-)-syringaresinol (6) was the most active compound with an IC50 value of 0.52 mM. It is noted that in zebrafish model, the known lignan 6 showed good in vivo antithrombotic effect with a value of 37% at a concentration of 30 µM, compared with the positive control aspirin with the inhibitory value of 74% at a concentration of 125µM. CONCLUSION: This study demonstrated that lignans, phenylpropanoid and biphenyl found in Piper wallichii may be responsible for antithrombotic effect of the titled plant.

Chemical constituents from Gnaphlium affine / 中草药

Objective: To investigate the chemical constituents from the stems of Gnaphlium affine. Methods: The constituents were isolated and purified by silica gel, Sephadex LH-20 column chromatography, and preparative TLC. The structures were identified on the basis of spectral data and physiochemical characteristics. Results: Fifteen compounds were isolated from 70% ethanol extracts of G. affine and identified as 3-methoxyphenol1-O-α-L-rhamnopyranosyl-(1→6)-O-β- D-glucopyranoside (1), 3', 5-dihydroxy-2-(4- hydroxybenzyl)-3-methoxybibenzyl (2), physcion (3), aldehyde (4), luteolin-4'-O-β-D-glucoside (5), faradiol 3-O-palmitate (6), betulinic acid (7), anabellamide (8), 4-O-D-glucopyranosyl-p-coumaric acid methyl ester (9), valene-1 (10)-ene-8, 11-diol (10), longumoside A (11), grossamiade K (12), quercetin-3-O-rutin-7-O-glucoside (13), luteolin-7-O-β-D-glucopyranosyl-(1→6)-[(6'''- O-caffeoylquinic)-β-D-glucopyranoside] (14), and isoverbascoside (15). Conclusion: Compounds 2 and 11 are reported from the plants in Gnaphalium L. for the first time. Compounds 2, 3, 6, 8, 13, and 14 are isolated from this plant material for the first time.

Chemical constituents from stems of Ficus auriculata / 中草药

Objective: To investigate the chemical constituents from the stems of Ficus auricalata. Methods: The constituents were isolated and purified by silica gel, Sephadex LH-20 column chromatography, and preparative TLC. Their structures were identified on the basis of spectral data and physiochemical characteristics. Results: Fourteen compounds were isolated from 95% ethanol extracts and identified as stigmast-4-ene-6β-ol-3-one (1), stigmast-4-ene-6α-ol-3-one (2), stigmast-4-ene-3-one (3), stigmast-5-ene-3β-ol-7-one (4), stigmast-4-ene-3, 6-dione (5), ergosterol peroxide (6), (20S)-3-oxo-hydroxytaraxastane (7), anabellamide (8), aurantiamide acetate (9) palmarumycin BG1 (10), 7-oxodehydroabietic acid (11), coniferyl aldehyde (12), S-(+)-dehydrovomifoliol (13), and 4-hydroxy-3-methoxy-benzoic acid (14). Conclusion: Compounds 2, 5, and 7-13 are reported from the plants in genus Ficus Linn. for the first time. All compounds are isolated from this plant material for the first time.

Chemical constituents of Syringae Cortex / 中国药学杂志

OBJECTIVE: To study the chemical constituents of Syringae Cortex. METHODS: The compounds were isolated and purified by silica (200-300), pre-HPLC and Sephadex LH-20 column chromatography. The structures were identified on the basis of spectral data and physiochemical properties. RESULTS: Twelve compounds were isolated and identified as syringin(I), sinapylal-dehyde 4-O-β-D-glucopyranoside(II), syringaldehyde(III), coniferyl aldehyde(IV), betulinic acid(V), oleuroprin(VI), oleoside dimethyl ester(VII), ligstroside(VIII), 2″-epifraxamoside (DC), β-sitosterol(X), n-triaconatanoic acid(XI) and 2-(4-hydroxyphe-nyl)-ethyl-l-dodecyloctadecanoate) (XII). CONCLUSION: Compounds II-V, VII and XI-XII are isolated from this plant for the first time.

Chemical constituents of Opuntia milpa (I) / 中草药

Objective: To investigate the chemical constituents of Opuntia milpa. Methods: The compounds were isolated by repeated silica gel chromatography and were elucidated by chemical and spectral analyses. Results: Ten compounds were isolated from the ethyl acelate extraction of O. milpa and identified as β-sitosterol (1), 1-heptadecanol (2), 5-hyadroxymemyl-2-furancarboxaldehyde (3), syringaldehyde (4), vanillin (5), p-hydroxybenzaldehyde (6), coniferyl aldehyde (7), p-hydroxyl-cinnamaldehyde (8), quercetin-3-methyl ether (9), and 2,6-dimethoxyphenol (10). Conclusion Compounds 3-10 are isolated from O. milpa for the first time.