Pingwah Tang

GC-MS-based metabolomics study of the responses to arachidonic acid in Blakeslea trispora.

The potential use of arachidonic acid (AA) to enhance the production of β-carotene in Blakeslea trispora was investigated in this work. To study the mechanism of the B. trispora response to AA, we used a systematic analytical approach to investigate the changes in the B. trispora cell metabolome at different time points after AA treatment. A maximum of β-carotene production was obtained when 0.4g/l AA was added after 36h of cultivation. Gas chromatography-mass spectrometry (GC-MS)-based metabolomics approach and a multivariate analysis were used to investigate the intracellular biochemical changes in B. trispora. With the aid of principal component analysis (PCA), the intracellular metabolite profiles of all the groups were distinguished. Moreover, a group classification and pairwise discrimination between the control and AA-treated groups were obtained through partial least-squares-discriminant analysis (PLS-DA), and 27 differential metabolites with variable importance in the projection (VIP) value higher than 1 were identified, which was also confirmed by the subsequent hierarchical cluster analysis (HCA). Separation of the control and AA-treated groups was mainly due to the compounds of the Krebs cycle, fatty acids and amino acids. With the treatment of AA, the glycolysis was enhanced and the use of glucose for fermentation was increased. The increased levels of some fatty acids and decreased levels of amino acids in the AA-treated cells could also be the responses to the addition of AA. Metabolomics provided a powerful methodology to gain insight in metabolic changes induced by metabolic stimulators in microorganisms.

Instability and Structural Change of 4-Methylsulfinyl-3-butenyl Isothiocyanate in the Hydrolytic Process.

Sulforaphene (4-methylsulfinyl-3-butenyl isothiocyanate), which has significant chemopreventive activities, is an important phytochemical ingredient produced by myrosinase hydrolysis of glucoraphenin (4-methylsulfinyl-3-butenyl glucosinolate) found in radish seeds. In this research, we found that sulforaphene was unstable and converted rapidly to a water-soluble degradation product in the hydrolytic process. The degradation product was successfully purified by preparative high-performance liquid chromatography on a C18 column using 10% methanol in water as the mobile phase. On the basis of MS and NMR spectroscopy data, the degradation product was identified to be 6-[(methylsulfinyl)methyl]-1,3-thiazinan-2-thione. The degradation pathway of sulforaphene was proposed in our study. Furthermore, low pH and metal ions were also found to have an effective inhibition to the degradation reaction of sulforaphene. Through adjusting the pH value of the system or adding metal ions after the content of sulforaphene has reached its maximum, the yield of sulforaphene increased significantly compared with that of the control.

Metabolic responses in Candida tropicalis to complex inhibitors during xylitol bioconversion.

During xylitol fermentation, Candida tropicalis is often inhibited by inhibitors in hemicellulose hydrolysate. The mechanisms involved in the metabolic responses to inhibitor stress and the resistances to inhibitors are still not clear. To understand the inhibition mechanisms and the metabolic responses to inhibitors, a GC/MS-based metabolomics approach was performed on C. tropicalis treated with and without complex inhibitors (CI, including furfural, phenol and acetic acid). Partial least squares discriminant analysis was used to determine the metabolic variability between CI-treated groups and control groups, and 25 metabolites were identified as possible entities responsible for the discrimination caused by inhibitors. We found that xylose uptake rate and xylitol oxidation rate were promoted by CI treatment. Metabolomics analysis showed that the flux from xylulose to pentose phosphate pathway increased, and tricarboxylic acid cycle was disturbed by CI. Moreover, the changes in levels of 1,3-propanediol, trehalose, saturated fatty acids and amino acids showed different mechanisms involved in metabolic responses to inhibitor stress. The increase of 1,3-propanediol was considered to be correlated with regulating redox balance and osmoregulation. The increase of trehalose might play a role in protein stabilization and cellular membranes protection. Saturated fatty acids could cause the decrease of membrane fluidity and make the plasma membrane rigid to maintain the integrity of plasma membrane. The deeper understanding of the inhibition mechanisms and the metabolic responses to inhibitors will provide us with more information on the metabolism regulation during xylitol bioconversion and the construction of industrial strains with inhibitor tolerance for better utilization of bioresource.

The stability and degradation kinetics of Sulforaphene in microcapsules based on several biopolymers via spray drying.

Sulforaphene (SFE) was extracted from the radish seeds and the purity of SFE extracted by our laboratory was 95%. It is well known that SFE can prevent cancers. It is also known that SFE is unstable to heat. To overcome the problem, SFE microcapsules using natural biopolymers were prepared by spray drying. The results indicated that SFE microcapsules using hydroxypropyl-β-cyclodextrin (HP-β-CD), maltodextrin (MD) and isolated soybean protein (SPI) as wall materials could effectively improve its stability against heat, especially SFE-loaded HP-β-CD and MD microcapsules. The amount of SFE in the microcapsules was found 20% higher than that of the non-encapsulated SFE under 90 °C in 168 h. Our finding suggested that the rate of degradation of the non-encapsulated and encapsulated SFE with HP-β-CD, MD and SPI followed the first-order kinetics. The speed of the degradation of the encapsulated SFE in biopolymers increased from SFE with HP-β-CD, to SFE with MD, and to SFE-SPI. The non-encapsulated SFE degrades fastest.

The stability and degradation mechanism of sulforaphene in solvents

Sulforaphene, a natural compound, has been investigated as a potential anticancer agent. However, the stability of sulforaphene, in various solvents, and its degradation pathway have not been appropriately reported. This instability impairs the preparation process, the biological evaluation experiments, and the applications of sulforaphene. In this study, the stability of sulforaphene stored at 26°C was investigated in each of the following six solvents: two kinds of protic solvents (methanol and ethanol) and four kinds of aprotic solvents (acetonitrile, dichloromethane, ethyl acetate and acetone). Sulforaphene was found to be stable in aprotic solvents and unstable in the protic solvents. The degradation products of sulforaphene in protic solvents (methanol and ethanol) were purified by the preparative HPLC and identified by ESI/MS and NMR ((1)H NMR). The degradation pathways of sulforaphene in methanol and ethanol were proposed. It was found that sulforaphene was degraded into two kinds of structural isomer in alcohols.

The mechanism of sulforaphene degradation to different water contents

Sulforaphene extracted from radish seeds was strongly associated with cancer prevention. However, sulforaphene was unstable in aqueous medium and at high temperature. This instability impairs many useful applications of sulforaphene. In this paper, the stability of sulforaphene (purity above 95%) during storage at -20°C, 4°C and 26°C was studied. The degradation product was purified by preparative HPLC and identified by ESI/MS, NMR ((1)H and (13)C NMR) and FTIR spectroscopy. The degradation pathway of sulforaphene was presented. Furthermore, we found that the degradation rate of sulforaphene was closely related to the water content of sulforaphene sample. The higher the water content was, the faster the sulforaphene sample degraded. A mathematical model was developed to predict the degradation constant at various water contents. It provided a guideline for industry to improve the stability of sulforaphene during preparation, application and storage.

Synergistic Effect of the Combination of Novel Suberoylanilide Hydroxamic Acid Derivatives with Cisplatin on Anti-proliferation of Human Cancer Cells

A novel, green, and atom-economical boric acid catalyzed direct amidation without the use of any coupling agents for the preparation of suberoylanilide hydroxamic acid (SAHA) and SAHA-based inhibitors targeting anti-proliferation of cancer cells is provided. The new SAHA-based inhibitor B123, when used alone, exhibited higher anti-proliferative activities than SAHA or Cisplatin against a number of human cancer cells. We have examined the effect of combination of these SAHA-based inhibitors with Cisplatin. We found synergistic effects of the combination of SAHA-based inhibitors with Cisplatin over a wide range of concentrations against human liver cancer cells HepG2 and two human lung cancer cell lines H1299 and H460. This synergism leads up to 8-fold of dose reduction for Cisplatin in the combination with our synthesized inhibitor B123 against H1299.

Metabolic regulation of α-linolenic acid on β-carotene synthesis in Blakeslea trispora revealed by a GC-MS-based metabolomic approach

Alpha-linolenic acid (ALA) is known for its ability to promote the production of β-carotene in Blakeslea trispora. However, the mechanism is still poorly understood. In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate analysis were used to study mechanisms underlying the regulation effects of ALA on β-carotene synthesis in B. trispora. ALA treatment promoted the biomass of B. trispora and β-carotene production. The maximum β-carotene production 5.344 mg L−1 was realized after 72 h of cultivation in the presence of 50 μL ALA. The intracellular metabolite profiles found upon treatment of the cells with different addition time points of ALA were unique and could be distinguished from the aid of principal component analysis (PCA). Furthermore, partial least-squares-discriminant analysis (PLS-DA) revealed a group classification and pairwise discrimination between the control and ALA treated groups, and 28 differential metabolites with variable importance in the projection (VIP) value greater than 1. The addition of ALA decreased the glycolysis, TCA cycle and fatty acid synthesis. The accumulation of linolenic acid and linoleic acid indicated that ALA was directly absorbed by the fungus and transformed into its own linolenic acid. As a result, the flux from acetyl-CoA to β-carotene synthesis increased. Besides, the addition of ALA increased the level of dissolved oxygen and the production of β-carotene.