Biotransformation of Bioactive Metabolites in Cassiae Semen by Endogenous Enzymes and Probiotics.

The present study evaluated the potential of endogenous enzymes and probiotics in transforming bioactive metabolites to reduce the purgative effect and improve the functional activity of Cassiae Semen and verified and revealed the biotransformation effect of endogenous enzymes. Although probiotics, especially Lactobacillus rhamnosus, exerted the transformation effect, the endogenous enzymes proved to be more effective in transforming the components of Cassiae Semen. After biotransformation by endogenous enzymes for 12 h, the levels of six anthraquinones in Cassiae Semen increased by at least 2.98-fold, and free anthraquinones, total phenolics, and antioxidant activity also showed significant improvement, accompanied by an 82.2% reduction in combined anthraquinones responsible for the purgative effect of Cassiae Semen. Further metabolomic analysis revealed that the biotransformation effect of endogenous enzymes on the bioactive metabolites of Cassiae Semen was complex and diverse, and the biotransformation of quinones and flavonoids was particularly prominent and occurred by three primary mechanisms, hydrolyzation, methylation, and dimerization, might under the action of glycosyl hydrolases, SAM-dependent methyltransferases, and CYP450s. Accordingly, biotransformation by endogenous enzymes emerges as a mild, economical, food safety risk-free, and effective strategy to modify Cassiae Semen into an excellent functional food.

Geraniin exerts cytoprotective effect against cellular oxidative stress by upregulation of Nrf2-mediated antioxidant enzyme expression via PI3K/AKT and ERK1/2 pathway.

BACKGROUND: Geraniin, an active compound with remarkable antioxidant activity, was isolated from Geranium sibiricum. The present study aimed to investigate whether geraniin has the ability to activate Nrf2, induce antioxidant enzyme expression and protect cells from oxidative damage. METHODS: The cells were pretreated with geraniin for 24h and exposed to hydrogen peroxide (H2O2) for 4h. Intracellular reactive oxygen species (ROS) levels, mitochondrial membrane potential and apoptosis were measured. We also investigated intracellular glutathione (GSH) levels and changes in nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated signaling cascade in cells treated with geraniin. RESULTS: We investigated the protective effects of geraniin against H2O2-induced apoptosis in HepG2 cells. Geraniin significantly reduced H2O2-induced oxidative damage in a dose dependent manner. Further, geraniin induced the expression of heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO1) and level of glutathione (GSH) in a concentration- and time-dependent manner, and increased Nrf2 nuclear translocation. The Nrf2-related cytoprotective effects of geraniin were PI3K/AKT and extracellular signal-regulated protein kinase1/2 (ERK1/2) pathway-dependent. However, inhibitors of PI3K/AKT and ERK1/2 (LY294002 or U0126) not only suppressed geraniin-induced nuclear translocation of Nrf2 but also abolished the expression of HO-1, NQO1 and GSH. CONCLUSIONS: These results demonstrated that geraniin induced Nrf2-mediated expression of antioxidant enzymes HO-1 and NQO1, presumably via PI3K/AKT and ERK1/2 signaling pathways, thereby protecting cells from H2O2-induced oxidative cell death. GENERAL SIGNIFICANCE: Geraniin, at least in part, offers an antioxidant defense capacity to protect cells from the oxidative stress-related diseases.

Establishment of hairy root cultures by Agrobacterium rhizogenes mediated transformation of Isatis tinctoria L. For the efficient production of flavonoids and evaluation of antioxidant activities.

In this work, Isatis tinctoria hairy root cultures (ITHRCs) were established as an alternative source for flavonoids (FL) production. I. tinctoria hairy root line V was found to be the most efficient line and was further confirmed by the PCR amplification of rolB, rolC and aux1 genes. Culture parameters of ITHRCs were optimized by Box-Behnken design (BBD), and eight bioactive FL constituents (rutin, neohesperidin, buddleoside, liquiritigenin, quercetin, isorhamnetin, kaempferol and isoliquiritigenin) were quali-quantitatively determined by LC-MS/MS. Under optimal conditions, the total FL accumulation of ITHRCs (24 day-old) achieved was 438.10 µg/g dry weight (DW), which exhibited significant superiority as against that of 2 year-old field grown roots (341.73 µg/g DW). Additionally, in vitro antioxidant assays demonstrated that ITHRCs extracts exhibited better antioxidant activities with lower IC50 values (0.41 and 0.39, mg/mL) as compared to those of field grown roots (0.56 and 0.48, mg/mL). To the best of our knowledge, this is the first report describing FL production and antioxidant activities from ITHRCs.

Variation in contents of main active components and antioxidant activity in leaves of different pigeon pea cultivars during growth.

Pigeon pea is an important and multiuse grain legume crop, and its leaves are a very valuable natural resource. To obtain a high-quality biological resource, it is necessary to choose the excellent cultivar and determine the appropriate harvest time. In this study, the variation in contents of main active components and antioxidant activity in leaves of six pigeon pea cultivars during growth were investigated. The level of each individual active component significantly varied during growth, but with a different pattern, and this variation was different among cultivars. Flavonoid glycosides orientin, vitexin, and apigenin-6,8-di-C-α-L-arabinopyranoside showed two peak values at mid-late and final stages of growth in most cases. Pinostrobin chalcone, longistyline C, and cajaninstilbene acid showed remarkablely higher values at the mid-late stage of growth than at other stages. Pinostrobin had an extremely different variation pattern compared to other active components. Its content was the highest at the earlier stage of growth. Principal component analysis (PCA) revealed that vitexin and apigenin-6,8-di-C-α-L-arabinopyranoside were mainly responsible for distinguishing cultivars analyzed. In a comprehensive consideration, the leaves should preferentially be harvested at the 135th day after sowing when the level of active components and antioxidant activity reached higher values. Cultivars ICP 13092, ICPL 87091, and ICPL 96053 were considered to be excellent cultivars with high antioxidant activity. Our findings can provide valuable information for producing a high-quality pigeon pea resource.

UV-induced changes of active components and antioxidant activity in postharvest pigeon pea [Cajanus cajan (L.) Millsp.] leaves.

In this study, the effect of UV irradiation (UV-A, UV-B, and UV-C) on phytochemicals, total phenolics, and antioxidant activity of postharvest pigeon pea leaves was evaluated. The response of pigeon pea leaves to UV irradiation was phytochemical specific. UV-B and UV-C induced higher levels of phytochemicals, total phenolics, and antioxidant activity in pigeon pea leaves compared with UV-A. Furthermore, UV-B irradiation proved to possess a long-lasting effect on the levels of phenolics and antioxidant activity. After adapting for 48 h at 4 °C following 4 h UV-B irradiation, total phenolics and antioxidant activity were approximately 1.5-fold and 2.2-fold increased from 39.4 mg GAE/g DM and 15.0 µmol GAE/g DM to 59.1 mg GAE/g DM and 32.5 µmol GAE/g DM, respectively. These results indicate that UV irradiation of pigeon pea leaves can be beneficial in terms of increasing active components and antioxidant activity.

Ionic liquid-based microwave-assisted extraction for the determination of flavonoid glycosides in pigeon pea leaves by high-performance liquid chromatography-diode array detector with pentafluorophenyl column.

In this study, an ionic liquid-based microwave-assisted extraction (ILMAE) followed by high-performance liquid chromatography-diode array detector with a pentafluorophenyl column for the extraction and quantification of eight flavonoid glycosides in pigeon pea leaves is described. Compared with conventional extraction methods, ILMAE is a more effective and environment friendly method for the extraction of nature compounds from herbal plants. Nine different types of ionic liquids with different cations and anions were investigated. The results suggested that varying the anion and cation had significant effects on the extraction of flavonoid glycosides, and 1.0 M 1-butyl-3-methylimidazolium bromide ([C4MIM]Br) solution was selected as solvent. In addition, the extraction procedures were also optimized using a series of single-factor experiments. The optimum parameters were obtained as follows: extraction temperature 60°C, liquid-solid ratio 20:1 mL/g and extraction time 13 min. Moreover, an HPLC method using pentafluorophenyl column was established and validated. Good linearity was observed with the regression coefficients (r(2)) more than 0.999. The limit of detection (LODs) (S/N = 3) and limit of quantification (LOQs) (S/N = 10) for the components were less than 0.41 and 1.47 µg/mL, respectively. The inter- and intraday precisions that were used to evaluate the reproducibility and relative standard deviation (RSD) values were less than 4.57%. The recoveries were between 97.26 and 102.69%. The method was successfully used for the analysis of samples of pigeon pea leaves. In conclusion, the developed ILMAE-HPLC-diode array detector using pentafluorophenyl column method can be applied for quality control of pigeon pea leaves and related medicinal products.