Using Porcine Jejunum Ex Vivo to Study Absorption and Biotransformation of Natural Products in Plant Extracts: Pueraria lobata as a Case Study

Herbal preparations (HPs) used in folk medicine are complex mixtures of natural products (NPs). Their efficacy in vivo after ingestion depends on the uptake of the active ingredient, and, in some cases, their metabolites, in the gastrointestinal tract. Thus, correlating bioactivities measured in vitro and efficacy in vivo is a challenge. An extract of Pueraria lobata rich in different types of isoflavones was used to evaluate the capacity of viable porcine small intestine ex vivo to elucidate the absorption of HP constituents, and, in some cases, their metabolites. The identification and transport of permeants across the jejunum was monitored by liquid chromatography-mass spectrometry (LC-MS), combining targeted and untargeted metabolite profiling approaches. It was observed that the C-glycoside isoflavones were stable and crossed the intestinal membrane, while various O-glycoside isoflavones were metabolized into their corresponding aglycones, which were then absorbed. These results are consistent with human data, highlighting the potential of using this approach. A thorough investigation of the impact of absorption and biotransformation was obtained without in vivo studies. The combination of qualitative untargeted and quantitative targeted LC-MS methods effectively monitored a large number of NPs and their metabolites, which is essential for research on HPs.

Controlled Fermentation Using Autochthonous Lactobacillus plantarum Improves Antimicrobial Potential of Chinese Chives against Poultry Pathogens

Chinese chives (CC) are rich in several antimicrobial constituents including organosulfur compounds, phenolics, and saponins, among others. Herein, we fermented CC juice using an autochthonous isolate, Lactobacillus plantarum having antimicrobial effects against poultry pathogens toward formulating an antimicrobial feed additive. Following 24 h of fermentation, the antimicrobial and antiviral activities of CC juice were significantly enhanced against poultry pathogens. However, the antioxidant activity of CC juice was significantly decreased following fermentation. Meanwhile, the compositional changes of CC juice following fermentation were also investigated. The total polyphenol, thiol, and allicin contents were significantly decreased in L. plantarum 24 h-fermented CC juice (LpCC) extract; however, total flavonoids increased significantly following fermentation. The untargeted metabolite profiling of nonfermented CC juice (NCC) and LpCC extracts was carried out using the ultra-high-performance liquid chromatography-linear trap quadrupole-orbitrap-tandem mass spectrometry (UHPLC-LTQ-Orbitrap-MS/MS) followed by multivariate analyses. The score plots of principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) based on UHPLC-LTQ-Orbitrap-MS/MS datasets displayed a clear segregation between the LpCC and NCC samples, which suggests their marked metabolomic disparity. Based on the multivariate analysis, we selected 17 significantly discriminant metabolites belonging to the different chemical classes including alkaloid, flavonols, saponins, fatty acids, amino acids, and organic acids. Notably, the flavonols including the glycosides of quercetin, kaempferol, and isorhamnetin as well as the saponins displayed significantly higher relative abundance in LpCC as compared with NCC. This study provides useful insights for the development of a fermented CC juice based antimicrobial feed additive to combat poultry infections.

Untargeted metabolite profiling on the water-soluble metabolites of edible bird's nest through liquid chromatography-mass spectrometry

Background and Aim: Edible bird's nest (EBN) is the nutrient-rich salivary bioproduct produced by swiftlets in Southeast Asia. Currently, researchers are exploring the therapeutic effects of EBN, such as cell growth promotion, antioxidant content, antiviral effects, bone strengthening, eyes care, and neuroprotection bioactivities. The therapeutic effects of EBN have been studied through different extraction methods but the metabolites profile of the EBN in each extract has not yet been elucidated. This study aimed to profile the water-soluble metabolites of EBN prepared in different extraction methods. Subsequently, an extraction method will be selected as an ideal extraction method for untargeted metabolite profiling on the water-soluble metabolites in EBN. Materials and Methods: In this study, water-soluble metabolites of EBN extracted by the four extraction methods were subjected to metabolite profiling through liquid chromatography-mass spectrometry (LC-MS). The extraction methods were acid extraction(ABN), pancreatic extraction (EzBN), eHMG extraction, and spray drying of HMG extraction (pHMG). The metabolite profiles, such as the number of metabolites and their identities in each extraction method, were evaluated through LC-MS analysis. Results: The identity of metabolites present in the four extraction methods is inconsistent. Based on LC-MS analysis, only one and six metabolites were extracted differently through EzBN and ABN, respectively, in the first pre-screening. Through the second LC-MS screening on pHMG and eHMG extraction methods, eHMG was selected as an ideal extraction method due to the highest numbers of water-soluble metabolites with an amount of 193 was detected. Besides, eHMG extraction method was able to extract sialic acid and a high percentage of secondary metabolites. Conclusion: This study suggests that eHMG is the ideal extraction method for extracting higher number of water-soluble metabolites from EBN and could be further developed as an extraction method for industry application. In addition, this study also has identified the types of primary and secondary metabolites present in EBN.

Early Pep-13-induced immune responses are SERK3A/B-dependent in potato

Potato plants treated with the pathogen-associated molecular pattern Pep-13 mount salicylic acid- and jasmonic acid-dependent defense responses, leading to enhanced resistance against Phytophthora infestans, the causal agent of late blight disease. Recognition of Pep-13 is assumed to occur by binding to a yet unknown plasma membrane-localized receptor kinase. The potato genes annotated to encode the co-receptor BAK1, StSERK3A and StSERK3B, are activated in response to Pep-13 treatment. Transgenic RNAi-potato plants with reduced expression of both SERK3A and SERK3B were generated. In response to Pep-13 treatment, the formation of reactive oxygen species and MAP kinase activation, observed in wild type plants, is highly reduced in StSERK3A/B-RNAi plants, suggesting that StSERK3A/B are required for perception of Pep-13 in potato. In contrast, defense gene expression is induced by Pep-13 in both control and StSERK3A/B-depleted plants. Altered morphology of StSERK3A/B-RNAi plants correlates with major shifts in metabolism, as determined by untargeted metabolite profiling. Enhanced levels of hydroxycinnamic acid amides, typical phytoalexins of potato, in StSERK3A/B-RNAi plants are accompanied by significantly decreased levels of flavonoids and steroidal glycoalkaloids. Thus, altered metabolism in StSERK3A/B-RNAi plants correlates with the ability of StSERK3A/B-depleted plants to mount defense, despite highly decreased early immune responses.

Untargeted Metabolite Profiling for Screening Bioactive Compounds in Digestate of Manure under Anaerobic Digestion

Untargeted metabolite profiling was performed on chicken manure (CHM), swine manure (SM), cattle manure (CM), and their respective digestate by XCMS coupled with MetaboAnalyst programs. Through global chemical profiling, the chemical characteristics of different digestates and types of manure were displayed during the anaerobic digestion (AD) process. As the feed for AD, CM had less easily-degradable organics, SM contained the least O-alkyls and anomerics of carbohydrates, and CHM exhibited relatively lower bio-stability. The derived metabolite pathways of different manure during the AD process were identified by MetaboAnalyst. Twelve, 8, and 5 metabolic pathways were affected by the AD process in CHM, SM, and CM, respectively. Furthermore, bioactive compounds of digestate were detected, such as amino acids (L-arginine, L-ornithine, L-cysteine, and L-aspartate), hormones (L-adrenaline, 19-hydroxy androstenedione, and estrone), alkaloids (tryptamine and N-methyltyramine), and vitamin B5, in different types of manure and their digestates. The combination of XCMS and MetaboAnalyst programs can be an effective strategy for metabolite profiling of manure and its anaerobic digestate under different situations.

Untargeted metabolomic analysis of the carotenoid-based orange coloration in Haliotis gigantea using GC-TOF-MS

Seafood coloration is typically considered an indicator of quality and nutritional value by consumers. One such seafood is the Xishi abalone (Haliotis gigantea), which displays muscle color polymorphism wherein a small subset of individuals display orange coloration of muscles due to carotenoid enrichment. However, the metabolic basis for carotenoid accumulation has not been thoroughly investigated in marine mollusks. Here, GC-TOF-MS-based untargeted metabolite profiling was used to identify key pathways and metabolites involved in differential carotenoid accumulation in abalones with variable carotenoid contents. Cholesterol was the most statistically significant metabolite that differentiated abalones with orange muscles against those with common white muscles. This observation is likely due to the competitive interactions between cholesterol and carotenoids during cellular absorption. In addition, the accumulation of carotenoids was also related to fatty acid contents. Overall, this study indicates that metabolomics can reflect physiological changes in organisms and provides a useful framework for exploring the mechanisms underlying carotenoid accumulation in abalone types.