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.

Evaluation of nutritional value, bioactivity and mineral content of quinoa bran in China and its potential use in the food industry.

Quinoa bran is a by-product during quinoa processing, which is not well used due to its high content of antinutritional factors. The nutritional, antinutritional, antioxidative and mineral content were analyzed in quinoa bran from five producing areas (Hebei, Shanxi, Qinghai, Inner Mongolia and Gansu Province) in China. The results showed that the mean values of protein, starch, fat, fiber, reducing sugar, ash, moisture and energy in quinoa bran were 9.35%, 47.37%, 8.26%, 10.74%, 3.68%, 6.25%, 9.29% and 360.2 kcal/100 g, respectively. Although the protein content in quinoa bran is lower than that in quinoa grain, it is comparable to that in other grains (rice, corn, millet and sorghum) and brans (wheat, oat and rice), so it has the commercial potential to be processed into animal feed or other edible food. The contents of antioxidant flavonoids (460.9 mg/100g) and polyphenols (477.8 mg/100 g) in quinoa bran were higher than those in quinoa grain, suggesting that quinoa bran had better antioxidant capacity. The contents of saponins, tannins and phytic acid in quinoa bran were 18.65, 0.30 and 0.73%, respectively. The content of saponins was nearly one times higher than that in quinoa grain, the contents of tannins and phytic acid, however, were lower than those in quinoa grain. Therefore, the removal of saponins is the key to eliminate the antinutritional properties of quinoa bran. The contents of macroelements (sodium, potassium, calcium, magnesium, phosphorus) and microelements (iron, manganese, copper, zinc, cobalt, molybdenum, selenium, barium) in quinoa bran were generally higher than those in quinoa grain, which was consistent with the results of ash determination. In summary, quinoa bran was found to be a rich source of nutritional and bioactive components and minerals. If the antinutritional problem can be overcome, quinoa bran has great potential for application in the food industry.

Adsorption and mechanistic study of the invasive plant-derived biochar functionalized with CaAl-LDH for Eu(III) in water.

Herein, we developed the invasive plant-derived biochar (IPB) functionalized with CaAl-LDH at five mass ratios using a physical mixture method, assessed their adsorption perform for Eu(III), and explored the relative mechanisms. Results show that the IPB successfully loaded CaAl-LDH in five composites and their Eu(III) sorption affinities were strongly affected by solution pH, contact time, temperature, and the mass ratio of LDH and IPB. All the sorpiton process for Eu(III) occurred on the heterogeneous surface of five composites and the boundary layer diffusion limited the chemical sorption rate. Interestingly, the CaAl-LDH/IPB composite with high ratio of IPB had higher sorption capacity than the one with high ratio of LDH due to larger porosity of the former. Three mechanisms containing ion exchange between Al and Eu ions, surface complexation with carboxyl- and oxygen-containing functional groups, and precipitation were involved in the Eu(III) sorption, but the dominant sorption mechanism for each CaAl-LDH/IPB composite differed with different mass ratio of CaAl-LDH and IPB. In composite with more IPB (e.g., CaAl-LDH/IPB-13), both ion exchange and surface complexes dominated the sorption process and the intensity of Eu3+ was identified with the one of Eu2O3. Whereas in composites with high LDH, ion exchange dominated the sorption and the intensity of Eu3+ was obviously higher than the one of Eu2O3. This research will provide a new perspective for the application of the LDH/biochar materials.

Activity of the antiestrogenic cajanin stilbene acid towards breast cancer.

Antiestrogenic therapy is a mainstay for estrogen receptor (ERα)-positive breast cancer. Due to the development of resistance to established antihormones such as tamoxifen, novel compounds are required. The low abundant cajanin stilbene acid (CSA) recently isolated by us from Pigeon Pea (Cajanus cajan) has structural similarities with estrogen. We analyzed the cytotoxic and anticancer activity of CSA in ERα-positive and -negative human breast cancer cells in vitro, in vivo and in silico. CSA exerts anticancer and antiestrogenic activities towards ERα-positive breast cancer, and it showed cytotoxicity towards tamoxifen-resistant MCF-7 cells, implying that CSA may be active against tamoxifen-resistant breast cancer cells. CSA showed low cytotoxicity in ERα-negative breast tumor cells as expected. Comparable cytotoxicity was observed towards p53 negative MCF-7 cells, implying that CSA is effective independent of the p53 status. Xenografted MCF-7 cells in nude mice were better inhibited by CSA than by cyclophosphamide. Testing of 8 primary cell cultures derived from human breast cancer biopsies showed that cell cultures from ER-positive tumors were more sensitive than from ER-negative ones. Dose-dependent decrease in ERα protein levels was observed upon CSA treatment. Synergistic effect with tamoxifen was observed in terms of increased p53 protein level. CSA affected pathways related to p53, cancer and cell proliferation. Gene promoter analyses supported the ERα regulation. CSA bound to the same site as 17ß-estradiol and tamoxifen on ERα. In conclusion, CSA exerts its anticancer effects in ERα-positive breast cancer cells by binding and inhibiting ERα.

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells.

BACKGROUND: The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet. MATERIAL AND METHODS: We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis. RESULTS: CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways. CONCLUSION: CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis.

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.

Simultaneous determination of phenolic compounds in Equisetum palustre L. by ultra high performance liquid chromatography with tandem mass spectrometry combined with matrix solid-phase dispersion extraction.

A method based on matrix solid-phase dispersion extraction followed by ultra high performance liquid chromatography with tandem mass spectrometry is presented for the extraction and determination of phenolic compounds in Equisetum palustre. This method combines the high efficiency of matrix solid-phase dispersion extraction and the rapidity, sensitivity, and accuracy of ultra high performance liquid chromatography with tandem mass spectrometry. The influential parameters of the matrix solid-phase dispersion extraction were investigated and optimized. The optimized conditions were as follows: silica gel was selected as dispersing sorbent, the ratio of silica gel to sample was selected to be 2:1 (400/200 mg), and 8 mL of 80% methanol was used as elution solvent. Furthermore, a fast and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the determination of nine phenolic compounds in E. palustre. This method was carried out within <6 min, and exhibited satisfactory linearity, precision, and recovery. Compared with ultrasound-assisted extraction, the proposed matrix solid-phase dispersion procedure possessed higher extraction efficiency, and was more convenient and time saving with reduced requirements on sample and solvent amounts. All these results suggest that the developed method represents an excellent alternative for the extraction and determination of active components in plant matrices.

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.