Feng-Lin Hu

Investigation on biochemical compositional changes during the microbial fermentation process of Fu brick tea by LC-MS based metabolomics.

Fu brick tea (FBT) is a unique post-fermented tea product which is fermented with fungi during the manufacturing process. In this study, we investigated the biochemical compositional changes occurring during the microbial fermentation process (MFP) of FBT based on non-targeted LC-MS, which was a comprehensive and unbiased methodology. Our data analysis took a two-phase approach: (1) comparison of FBT with other tea products using PCA analysis to exhibit the characteristic effect of MFP on the formation of Fu brick tea and (2) comparison of tea samples throughout the MFP of FBT to elucidate the possible key metabolic pathways produced by the fungi. Non-targeted LC-MS analysis clearly distinguished FBT with other tea samples and highlighted some interesting metabolic pathways during the MFP including B ring fission catechin. Our study demonstrated that those fungi had a significant influence on the biochemical profiles in the FBT and consequently contributed to its unique quality.

EGCG inhibit chemical reactivity of iron through forming an Ngal-EGCG-iron complex.

Accumulated evidence indicates that the interconversion of iron between ferric (Fe3+) and ferrous (Fe2+) can be realized through interaction with reactive oxygen species in the Fenton and Haber–Weiss reactions and thereby physiologically effects redox cycling. The imbalance of iron and ROS may eventually cause tissue damage such as renal proximal tubule injury and necrosis. Many approaches were exploited to ameliorate the oxidative stress caused by the imbalance. (−)-Epigallocatechin-3-gallate, the most active and most abundant catechin in tea, was found to be involved in the protection of a spectrum of renal injuries caused by oxidative stress. Most of studies suggested that EGCG works as an antioxidant. In this paper, Multivariate analysis of the LC–MS data of tea extracts and binding assays showed that the tea polyphenol EGCG can form stable complex with iron through the protein Ngal, a biomarker of acute kidney injury. UV–Vis and Luminescence spectrum methods showed that Ngal can inhibit the chemical reactivity of iron and EGCG through forming an Ngal–EGCG–iron complex. In thinking of the interaction of iron and ROS, we proposed that EGCG may work as both antioxidant and Ngal binding siderphore in protection of kidney from injuries.

Metabolic effect of an exogenous gene on transgenic beauveria bassiana using liquid chromatography-mass spectrometry-based metabolomics

Genetic modification of Beauveria bassiana with the scorpion neurotoxin aaIT gene can distinctly increase its insecticidal activity, whereas the effect of this exogenous gene on the metabolism of B. bassiana is unknown until now. Thus, we investigate the global metabolic profiling of mycelia and conidia of transgenic and wild-type B. bassiana by liquid chromatography-mass spectrometry (LC-MS). Principal component analysis (PCA) and orthogonal projection to latent structure discriminant analysis (OPLS-DA) reveal clear discrimination of wild-type mycelia and conidia from transgenic mycelia and conidia. The decrease of glycerophospholipids, carnitine, and fatty acids and the increase of oxylipins, glyoxylate, pyruvic acid, acetylcarnitine, fumarate, ergothioneine, and trehalose in transgenic mycelia indicate the enhanced oxidative reactions. In contrast, most metabolites related to oxidative stress are not altered significantly in conidia, which implies that there will be no significant oxidative stress reaction when the aaIT gene is quiescent in cells.

Differential metabolic responses of Beauveria bassiana cultured in pupae extracts, root exudates and its interactions with insect and plant.

Beauveria bassiana is a kind of world-wide entomopathogenic fungus and can also colonize plant rhizosphere. Previous researches showed differential expression of genes when entomopathogenic fungi are cultured in insect or plant materials. However, so far there is no report on metabolic alterations of B. bassiana in the environments of insect or plant. The purpose of this paper is to address this problem. Herein, we first provide the metabolomic analysis of B. bassiana cultured in insect pupae extracts (derived from Euproctis pseudoconspersa and Bombyx mori, EPP and BMP), plant root exudates (derived from asparagus and carrot, ARE and CRE), distilled water and minimal media (MM), respectively. Principal components analysis (PCA) shows that mycelia cultured in pupae extracts and root exudates are evidently separated and individually separated from MM, which indicates that fungus accommodates to insect and plant environments by different metabolic regulation mechanisms. Subsequently, orthogonal projection on latent structure-discriminant analysis (OPLS-DA) identifies differential metabolites in fungus under three environments relative to MM. Hierarchical clustering analysis (HCA) is performed to cluster compounds based on biochemical relationships, showing that sphingolipids are increased in BMP but are decreased in EPP. This observation further implies that sphingolipid metabolism may be involved in the adaptation of fungus to different hosts. In the meantime, sphingolipids are significantly decreased in root exudates but they are not decreased in distilled water, suggesting that some components of the root exudates can suppress sphingolipid to down-regulate sphingolipid metabolism. Pathway analysis finds that fatty acid metabolism is maintained at high level but non-ribosomal peptides (NRP) synthesis is unaffected in mycelia cultured in pupae extracts. In contrast, fatty acid metabolism is not changed but NRP synthesis is high in mycelia cultured in root exudates and distilled water. This indicates that fungal fatty acid metabolism is enhanced when contacting insect, but when in the absence of insect hosts NRP synthesis is increased. Ornithine, arginine and GABA are decreased in mycelia cultured in pupae extracts and root exudates but remain unchanged in distilled water, which suggests that they may be associated with fungal cross-talk with insects and plants. Trehalose and mannitol are decreased while adenine is increased in three conditions, signifying carbon shortage in cells. Together, these results unveil that B. bassiana has differential metabolic responses in pupae extracts and root exudates, and metabolic similarity in root exudates and distilled water is possibly due to the lack of insect components.

Comparison of cytotoxic extracts from fruiting bodies, infected insects and cultured mycelia of Cordyceps formosana

A resazurin method was employed to test and compare cytotoxicity of extracts from fruiting bodies, insects and cultured mycelia of Cordyceps formosana against Chinese hamster ovary (CHO) cells. Results showed that the cultured mycelia had much stronger cytotoxicity than that of the fruiting bodies and infected insects. This suggests that using cultured mycelia to substitute a natural Cordyceps may result in poisoning. A combined method of HPLC-PAD-HRMS and cytotoxic analysis revealed that the most toxic compound (Compound 1) was found mainly in the cultured mycelia and also a small amount in the infected insect body of the Cordyceps, but not in the fruiting body. The second toxic compound (Compound 2) was found in all structures of Cordyceps and in cultured mycelia. Different contents of the toxic compounds resulted in the different cytotoxicity of the extracts. Compound 1 and Compound 2 were prepared with preparative HPLC as yellow and orange powders, respectively. Cytotoxic tests showed that the median lethal dose (LD₅₀) against CHO cells of Compound 1 was 18.3 ± 0.2 and 103.7 ± 5.9 μg/mL for Compound 2. Compound 1 and Compound 2 were identified as rugulosin and skyrin by HRMS, UV and NMR data. The two compounds were never previously isolated from the genera Cordyceps and Hirsutella and their cytotoxicity against CHO cells was also reported for the first time.

Novel acetylcholinesterase inhibitors from Zijuan tea and biosynthetic pathway of caffeoylated catechin in tea plant

Zijuan tea is a special cultivar of Yunnan broad-leaf tea (Camellia sinensis var. assamica) with purple buds, leaves, and stems. Phytochemical study on this tea led to the discovery of three hydroxycinnamoylated catechins (HCCs) (1-3), seven other catechins (4-10), three proanthocyanidins (11-13), five flavones and flavone glycosides (14-18), two alkaloids (19, 20), one steroid (21), and one phenylpropanoid glycoside (22). The isolation and structural elucidation of the caffeoylated catechin (1) by means of spectroscopic techniques were described. We also provide the first evidence that 1 is synthesized via a two-step pathway in tea plant. The three HCCs (1-3) were investigated on their bioactivity through molecular modeling simulation and biochemical experiments. Our results show that they bind acetylcholinesterase (AChE) tightly and have strong AChE inhibitory activity with IC50 value at 2.49, 11.41, 62.26μM, respectively.

New tyrosinase inhibitors from Paecilomyces gunnii.

Through screening 50 strains of entomopathogenic fungi and rescreening of 7 strains of Paecilomyces gunnii, a methanol extract of liquid-cultivated mycelia of P. gunnii was found to have the strongest tyrosinase inhibitory activity. Preparative high-speed counter-current chromatography (HSCCC) guided by high-performance liquid chromatography (HPLC)-electrospray ionization (ESI)-high-resolution mass spectrometry (HRMS) was employed for the isolation and purification of the active components, and three new compounds with half inhibition concentration (IC50) of 0.11, 0.17, and 0.14 mM against diphenolase were obtained from the extract, respectively. Their chemical structures were identified by HRMS, one- and two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy as paecilomycones A, B, and C. Structure and activity studies showed that the tyrosinase inhibition activities are positively related to the number of hydroxyl groups on the paecilomycones.

Metabolic responses of Beauveria bassiana to hydrogen peroxide-induced oxidative stress using an LC-MS-based metabolomics approach.

The entomopathogenic fungus, Beauveria bassiana, is commonly used as a biological agent for pest control. Environmental and biological factors expose the fungus to oxidative stress; as a result, B. bassiana has adopted a number of anti-oxidant mechanisms. In this study, we investigated metabolites of B. bassiana that are formed in response to oxidative stress from hydrogen peroxide (H2O2) by using a liquid chromatography mass spectrometry (LC-MS) approach. Partial least-squares discriminant analysis (PLS-DA) revealed differences between the control and the H2O2-treated groups. Hierarchical cluster analysis (HCA) showed 18 up-regulated metabolites and 25 down-regulated metabolites in the H2O2-treated fungus. Pathway analysis indicated that B. bassiana may be able to alleviate oxidative stress by enhancing lipid catabolism and glycometabolism, thus decreasing membrane polarity and preventing polar H2O2 or ROS from permeating into fungal cells and protecting cells against oxidative injury. Meanwhile, most of the unsaturated fatty acids that are derived from glycerophospholipids hydrolysis can convert into oxylipins through autoxidation, which can prevent the reactive oxygen of H2O2 from attacking important macromolecules of the fungus. Results showed also that H2O2 treatment can enhance mycotoxins production which implies that oxidative stress may be able to increase the virulence of the fungus. In comparison to the control group, citric acid and UDP-N-acetylglucosamine were down-regulated, which suggested that metabolic flux was occurring to the TCA cycle and enhancing carbohydrate metabolism. The findings from this study will contribute to the understanding of how the molecular mechanisms of fungus respond to environmental and biological stress factors as well as how the manipulation of such metabolisms may lead to selection of more effective fungal strains for pest control.

A New Fatty Acid from the Leaves and Stems of Clerodendron trichotomum

A new fatty acid, (5E,7E)-9-oxooctadeca-5,7-dienoic acid (1), was isolated from the leaves and stems of Clerodendron trichotomum. The structure of the new compound was elucidated by analysis of spectrometric data.