Metabolomics Reveals Rubiadin Accumulation and the Effects of Methyl Jasmonate Elicitation in Damnacanthus major Calli.

Callus suspension techniques have been considered attractive for improving bioactive metabolite productivity; methyl jasmonate (MeJA) is a widely used elicitor for stimulating synthetic pathways. In this study, a multivariate analysis-based metabolomics approach was employed to investigate the primary and specialized metabolites in the leaves, unelicited calli, and 100 or 200 µM MeJA elicited calli of Damnacanthus major. Rubiadin, a powerful anthraquinone with various therapeutic properties, was only identified in D. major calli, accumulating in a MeJA elicitation concentration-dependent manner. Callus cultures also contained high levels of amino acids, sugars, and phenolic compounds, indicating energy metabolism and metabolic adaptation responses for proliferation and stabilization. Regarding MeJA application, elicited calli contained higher amounts of quinic acid, kaempferol, and glucose with lower amounts of sucrose and raffinose than those in the unelicited control, which were closely related to protective mechanisms against MeJA. Moreover, excessive elicitation increased the asparagine, fructose, and raffinose levels and decreased the glucose and sucrose levels, which was ascribed to increased activation of the aminoacyl-tRNA biosynthesis pathway and wider utilization of glucose than of fructose after sucrose degradation. These results will be useful for optimizing plant cell culture techniques to achieve high production rates for valuable specialized metabolites.

Proliferation and Metabolic Profiling of Cynanchum wilfordii Adventitious Roots Using Explants from Different Cultivation Methods.

Cynanchum wilfordii root is used in traditional herbal medicine owing to its various pharmacological activities. However, C. wilfordii roots are misused owing to their morphological similarities with C. auriculatum. Adventitious root (AR) culture can prevent such misuse, and the selection of plant materials is an important procedure for producing high-quality ARs. This study aimed to compare the proliferation and metabolic profiles of C. wilfordii ARs in two types of explants from different cultivation methods (either cultivated in open field (ECF) or cultivated on a heap of C. wilfordii (ECH)). After 4 weeks of culture, the proliferation rate and number and length of secondary ARs were determined, and 3/4 Murashige and Skoog (MS) salt medium, 4.92 µM indole-3-butyric acid (IBA), and 5% sucrose were suggested as the best proliferation conditions for ARs originating from both ECF and ECH. Through metabolic profiling, ARs from ECH were found to show higher accumulation patterns for flavonoids, polysaccharides, hydroxyacetophenones, aromatic amino acids, and mono-unsaturated fatty acids, which were ascribed to the activation of flavonoid biosynthesis, the phenylpropanoid pathway, and fatty acid desaturase, stimulated by abiotic stresses. In contrast, ARs from ECF had higher levels of TCA cycle intermediates, amino acids in the aspartate-glutamate pathway, and saturated and polyunsaturated fatty acids, indicating energy metabolism and plant development. Overall, the current study provided information on the optimal conditions for inducing C. wilfordii ARs with higher amounts of bioactive compounds.

Oxidative Stress Amplifying Polyprodrug Micelles as Drug Carriers for Combination Anticancer Therapy.

Cancer cells are more vulnerable to reactive oxygen species (ROS)-mediated oxidative stress than normal cells due to disturbed redox balance. It can be postulated that ROS-generating drug carriers exert anticancer actions, leading to combination anticancer therapy with drug payloads. Here, we report a ROS-generating polyprodrug of cinnamaldehyde (CA) that not only serves as a drug carrier but also synergizes with drug payloads. The polyprodrug of CA (pCA) incorporates ROS-generating CA in the backbone of an amphiphilic polymer through an acid-cleavable acetal linkage. pCA could self-assemble with tumor-targeting lipopeptide (DSPE-PEG-RGD) and encapsulate doxorubicin (DOX) to form T-pCAD micelles. At acidic pH, T-pCAD micelles release both CA and DOX to exert synergistic anticancer actions. Animal studies using mouse xenograft models revealed that T-pCAD micelles accumulate in tumors preferentially and suppress the tumor growth significantly. Based on the oxidative stress amplification and acid-responsiveness, ROS-generating pCAD micelles hold tremendous potential as drug carriers for combination anticancer therapy.

Gene Expression and Metabolic Analyses of Nontransgenic and AtPAP1 Transgenic Tobacco Infected with Potato Virus X (PVX).

Potato virus X (PVX), a species of the genus Potexvirus, is a plant pathogenic virus that causes severe symptoms such as mild mosaic, crinkling, necrosis, and mottling on leaves. The objectives of the present study were to investigate the effect of PVX virus infection on the metabolic system in nontransgenic and Arabidopsis thaliana production of anthocyanin pigment 1 (AtPAP1) transgenic tobacco using transcript expression analysis and metabolic profiling. Potato virus X inoculation increased the gene expression of phenylpropanoid and flavonoid biosynthesis and the production of chlorogenic acid, p-coumaric acid, benzoic acid, rutin, quercetin, and kaempferol in nontransgenic tobacco leaves. However, in the AtPAP1 transgenic tobacco leaves, PVX inoculation decreased the expression of AtPAP1 and phenylpropanoid and flavonoid biosynthesis genes, and the production of phenolics and anthocyanin also declined. In contrast, the levels of amino acids and tricarboxylic acid (TCA) cycle intermediates increased after infection in the AtPAP1 transgenic plant leaves. To date, these results have not been reported previously. We suggest that PVX infection decreases AtPAP1 expression, leading to the downregulation of phenylpropanoid and flavonoid biosynthesis in transgenic plants.

Clinical Evidence of Effects of Green Mandarin (Putgyul) Extract on Skin Aging: A Randomized, Double Blind, Placebo-Controlled Study.

Green mandarins are widely consumed unripe as mandarin oranges (Citrus unshiu Marcov.), which exhibit anti-inflammatory and anti-wrinkle effects by inhibiting the production of inflammatory cytokines and matrix metalloproteinase. A randomized, double-blind, placebo-controlled clinical study was performed to verify the skin improvement efficacy and safety of green mandarin extract (PTE). For the standardization of PTE, narirutin was set as a marker compound, and PTE with a constant narirutin content was prepared for the study. After randomizing subjects with periorbital wrinkles, they were orally administered PTE (300 mg/day) or a placebo for 12 weeks. Periorbital wrinkles were measured using PRIMOSCR SF. Skin elasticity, moisture content, transepidermal water loss, and gloss were also measured. In the study results, the depth, volume, and skin roughness of the periorbital wrinkles were significantly improved compared to the control group (p = 0.011, 0.009, and 0.004, respectively). The survey confirmed that the skin condition improved after PTE consumption for 12 weeks. No adverse reactions associated with PTE were observed during the study period. Thus, the results demonstrate that PTE effectively improves UV-induced skin wrinkles. Therefore, it is considered that PTE has sufficient value as a functional food ingredient that can prevent skin aging.

Metabolic profiling and antioxidant activity during flower development in Agastache rugosa.

Our previous study showed that flowers of Agastache rugosa had higher phenolic levels and higher antibacterial and antioxidant capacity compared to those of the leaves and stems. The aim of this study was to provide information on the variation in primary and secondary metabolites during flower development in A. rugosa by using high performance liquid chromatography (HPLC) and assays of total anthocyanin (TAC), flavonoid (TFC), and phenolic content (TPC), as well as gas chromatography time-of-flight mass spectrometry (GC-TOFMS) analysis. Assays of TPC, TAC, and TFC showed that the floral bud (stage I) contained higher TPC than did the partially open flower (stage II) and fully open flower (stage III). However, the TFC was the highest at stage II, and the highest TAC was observed at stage III. Furthermore, HPLC analysis revealed that the level of total phenylpropanoids, including rosmarinic acid, tilianin, acacetin, 4-hydroxybenzoic acid, caffeic acid, chlorogenic acid, trans-cinnamic acid, rutin, (-)-epicatechin, quercetin, and kaempferol, was higher in stages I and II, but the concentrations of rutin and rosmarinic acid were highest in stage III. A total of 43 compounds, including amino acids, organic acids, phenolic compounds, sugars, photorespiration-related compounds, and intermediates of the tricarboxylic acid cycle, were identified through GC-TOFMS analysis. Of these compounds, most amino acids decreased during flower development. In contrast, the increase in concentrations of glucose and sucrose were observed from stages I to III. In this study, health-beneficial compounds were identified and quantified in flowers of A. rugosa. Accordingly, our results suggests that A. rugosa flowers can potentially be used as biomaterials for pharmaceuticals, cosmetics, food, and related industries. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s12298-021-00945-z).

Profiles of Essential Oils and Correlations with Phenolic Acids and Primary Metabolites in Flower Buds of Magnolia heptapeta and Magnolia denudata var. purpurascens.

Magnolia flower buds are a source of herbal medicines with various active compounds. In this study, differences in the distribution and abundance of major essential oils, phenolic acids, and primary metabolites between white flower buds of Magnolia heptapeta and violet flower buds of Magnolia denudata var. purpurascens were characterised. A multivariate analysis revealed clear separation between the white and violet flower buds with respect to primary and secondary metabolites closely related to metabolic systems. White flower buds contained large amounts of monoterpene hydrocarbons (MH), phenolic acids, aromatic amino acids, and monosaccharides, related to the production of isoprenes, as MH precursors, and the activity of MH synthase. However, concentrations of ß-myrcene, a major MH compound, were higher in violet flower buds than in white flower buds, possibly due to higher threonine levels and low acidic conditions induced by comparatively low levels of some organic acids. Moreover, levels of stress-related metabolites, such as oxygenated monoterpenes, proline, and glutamic acid, were higher in violet flower buds than in white flower buds. Our results support the feasibility of metabolic profiling for the identification of phytochemical differences and improve our understanding of the correlated biological pathways for primary and secondary metabolites.

Imaging and Targeted Antibacterial Therapy Using Chimeric Antimicrobial Peptide Micelles.

Infectious diseases induced by multidrug-resistant bacteria are a challenging problem in medicine because of global rise in the drug resistance to pathogenic bacteria. Despite great efforts on the development of antibiotics and antimicrobial agents, there is still a great need to develop a strategy to early detect bacterial infections and eradicate bacteria effectively and simultaneously. The innate immune systems of various organisms produce antimicrobial peptides, which kill a broad range of bacteria with minimal cytotoxicity to mammalian cells. Therefore, antimicrobial peptides have recently attracted increasing attention as an alternative to conventional antibiotics in antibacterial medications. Here, we report a new family of antibacterial agents, which is formulated from self-assembly of a chimeric antimicrobial lipopeptide (DSPE-HnMc) and amphiphilic biodegradable polymers. HnMc micelles could effectively bind the bacterial membrane to kill a wide spectrum of bacteria and bacterial biofilms. In the studies of mouse models of drug-resistant bacterial infections, HnMc micelles could target bacterial infections with high specificity and also kill drug-resistant bacteria effectively, demonstrating the great potential of HnMc micelles as imaging and targeted antibacterial agents. These findings also provide new insight into the design of antimicrobial peptide-based nanomedicine for detection and treatment of bacterial infections.

Integrated Proteomics and Metabolomics Analysis Highlights Correlative Metabolite-Protein Networks in Soybean Seeds Subjected to Warm-Water Soaking.

Soaking of soybean seeds is a prerequisite for the production of soy foods, and it has been shown that the extent of water absorbed during different imbibition conditions directly affects the quality of the subsequent soybean seed products by yet unknown mechanisms. In order to elucidate the molecular changes in soybean seeds during different soaking temperatures, we performed an integrated proteomics and metabolomics analysis of seeds soaked at 4, 25, and 55 °C. Proteomics analysis revealed that various enzymes related to carbohydrate and protein hydrolysis were activated in soybean seeds during water soaking at 55 °C. Interestingly, results obtained from this integrated proteomics and metabolomics study showed changes in various metabolites, including isoflavones, amino acids, and sugars, that were positively correlated with proteome changes occurring upon soaking at 55 °C. Furthermore, soaking of soybean seeds at 55 °C resulted in degradation of indigestible anti-nutrients such as raffinose oligosaccharides. Taken together, our results suggest that the seed soaking at a high temperature (55 °C) increases the nutritional value of soybean seeds by decreasing the contents of some of the common anti-nutrients.

Comparative metabolic profiling of cultivated and wild black soybeans reveals distinct metabolic alterations associated with their domestication.

Generally, cultivated black soybean (CBS) has been used as a major source of various nutrients for humans and animals. To assess the metabolic alterations induced by domestication in soybean, we performed a comprehensive metabolite profiling of 56 soybean varieties, including 28 CBS and 28 wild black soybean (WBS) varieties. A total of 48 metabolites were characterized, including 45 primary and 3 secondary metabolites, from CBS and WBS. The results of principal component analysis and hierarchical cluster analysis (HCA) revealed significant metabolic differences between CBS and WBS that were closely related to metabolic pathways. The results indicate that flavonoids correlated positively with phenylalanine, a precursor for phenylpropanoid biosynthesis; the contents of flavonoids and phenylpropanoids were higher in WBS. Pathway analysis revealed that CBS contained large amounts of TCA cycle intermediates, amino acids, and fatty acids as a result of increased energy metabolism, amino acid metabolism, and seed filling. The projection to latent structure method, using the partial least squares method, was applied to predict the flavonoid content in soybean seed, which indicated that sucrose, threonic acid, citric acid, and fatty acids are important in predicting the antioxidant content of samples. This work will provide important information for designing new soybean cultivars with enhanced nutritional and agricultural traits.

A high-throughput platform for interpretation of metabolite profile data from pepper (Capsicum) fruits of 13 phenotypes associated with different fruit maturity states.

Nowadays, novel tools have been developed for efficient analysis and visualization of large-scale metabolite profile data associated with metabolic pathways. A high-throughput platform using PathVisio 3 combined with multivariate analysis is proposed for the first time. Additionally, this is the first analysis of the relationships among terpenoids monoterpene, sesquiterpene, triterpene, and tetraterpene during pepper fruit ripening, and their changes. This platform was successfully applied to interpret large-scale data related to 131 metabolites from mature and immature fruits of 13 pepper phenotypes. The carotenoid-derived volatiles, such as dihydroactinidiolide and ß-ionone were closely correlated with carotenoids, indicating that the synthesis and degradation of carotenoids occurred in pepper fruit mature stage. Using PathVisio 3, the metabolic changes in pathway could be presented quickly, revealing the accumulation of stress-related metabolites, such as proline, capsaicin, and phenylalanine, in the mature stage. This approach could provide useful information about comprehensive biochemical regulation of fruit ripening.

Metabolic Profiling-Based Evaluation of the Fermentative Behavior of Aspergillus oryzae and Bacillus subtilis for Soybean Residues Treated at Different Temperatures.

Soybean processing, e.g., by soaking, heating, and fermentation, typically results in diverse metabolic changes. Herein, multivariate analysis-based metabolic profiling was employed to investigate the effects of fermentation by Aspergillus oryzae or Bacillus subtilis on soybean substrates extracted at 4, 25, or 55 °C. As metabolic changes for both A. oryzae and B. subtilis were most pronounced for substrates extracted at 55 °C, this temperature was selected to compare the two microbial fermentation strategies, which were shown to be markedly different. Specifically, fermentation by A. oryzae increased the levels of most organic acids, γ-aminobutyric acid, and glutamine, which were ascribed to carbohydrate metabolism and conversion of glutamic acid into GABA and glutamine. In contrast, fermentation by B. subtilis increased the levels of most amino acids and isoflavones, which indicated the high activity of proteases and ß-glucosidase. Overall, the obtained results were concluded to be useful for the optimization of processing steps in terms of nutritional preferences.

Acid-activatable polymeric curcumin nanoparticles as therapeutic agents for osteoarthritis.

Curcumin, a primary active element of turmeric, has potent antioxidant and anti-inflammatory activity, but its low bioavailability is a major hurdle in its pharmaceutical applications. To enhance the therapeutic efficacy of curcumin, we exploited polymeric prodrug strategy. Here, we report rationally designed acid-activatable curcumin polymer (ACP), as a therapeutic prodrug of curcumin, in which curcumin was covalently incorporated in the backbone of amphiphilic polymer. ACP could self-assemble to form micelles that rapidly release curcumin under the acidic condition. The potential of ACP micelles as therapeutics for osteoarthritis was evaluated using a mouse model of monoidoacetic acid (MIA)-induced knee osteoarthritis. ACP micelles drastically protected the articular structures from arthritis through the suppression of tumor necrosis factor-alpha (TNF-α) and interleukin 1ß (IL-1ß). Given their pathological stimulus-responsiveness and potent antioxidant and anti-inflammatory activities, ACP micelles hold remarkable potential as a therapeutic agent for not only osteoarthritis but also various inflammatory diseases.

Hydrogen peroxide-activatable polymeric prodrug of curcumin for ultrasound imaging and therapy of acute liver failure.

Curcumin is a major active phenolic component of turmeric and has gained great attention in pharmaceutics due to its potent antioxidant, anti-inflammatory and anticancer activity. Here, we developed poly(oxalate-co-curcumin) (POC) as a hydrogen peroxide (H2O2)-activatable polymeric prodrug of curcumin by incorporating curcumin in the backbone of H2O2-responsive polyoxalate. POC particles effectively scavenged H2O2 and released curcumin in a H2O2-triggered manner. POC particles exhibited excellent antioxidant and anti-inflammatory activity in activated cells. POC particles intravenously administrated into acetaminophen-intoxicated mice remarkably suppressed the level of alanine transaminase and inhibited apoptotic cell death in liver. Interestingly, POC particles could also enhance the ultrasound contrast in the intoxicated liver due to CO2 bubble generation through H2O2-triggered oxidation of peroxalate esters. Given their H2O2-responsiveness and highly potent antioxidant activity, POC particles hold great translational potential as theranostic agents for H2O2-associated diseases.