Characterization of effective phytochemicals in traditional Chinese medicine by mass spectrometry.

Traditional Chinese medicines (TCMs) have been widely used in clinical and healthcare applications around the world. The characterization of the phytochemical components in TCMs is very important for studying the therapeutic mechanism of TCMs. In the analysis process, sample preparation and instrument analysis are key steps to improve analysis performance and accuracy. In recent years, chromatography combined with mass spectrometry (MS) has been widely used for the separation and detection of trace components in complex TCM samples. This article reviews various sample preparation techniques and chromatography-MS techniques, including the application of gas chromatography-MS and liquid chromatography-MS and other MS techniques in the characterization of phytochemicals in TCM materials and Chinese medicine products. This article also describes a new ambient ionization MS method for rapid and high-throughput analysis of TCM components.

Long, Noncoding RNA SRA Induces Apoptosis of ß-Cells by Promoting the IRAK1/LDHA/Lactate Pathway.

Long non-coding RNA steroid receptor RNA activators (LncRNA SRAs) are implicated in the ß-cell destruction of Type 1 diabetes mellitus (T1D), but functional association remains poorly understood. Here, we aimed to verify the role of LncRNA SRA regulation in ß-cells. LncRNA SRAs were highly expressed in plasma samples and peripheral blood mononuclear cells (PBMCs) from T1D patients. LncRNA SRA was strongly upregulated by high-glucose treatment. LncRNA SRA acts as a microRNA (miR)-146b sponge through direct sequence-structure interactions. Silencing of lncRNA SRA increased the functional genes of Tregs, resulting in metabolic reprogramming, such as decreased lactate levels, repressed lactate dehydrogenase A (LDHA)/phosphorylated LDHA (pLDHA at Tyr10) expression, decreased reactive oxygen species (ROS) production, increased ATP production, and finally, decreased ß-cell apoptosis in vitro. There was a positive association between lactate level and hemoglobin A1c (HbA1c) level in the plasma from patients with T1D. Recombinant human interleukin (IL)-2 treatment repressed lncRNA SRA expression and activity in ß-cells. Higher levels of lncRNA-SRA/lactate in the plasma are associated with poor regulation in T1D patients. LncRNA SRA contributed to T1D pathogenesis through the inhibition of miR-146b in ß-cells, with activating signaling transduction of interleukin-1 receptor-associated kinase 1 (IRAK1)/LDHA/pLDHA. Taken together, LncRNA SRA plays a critical role in the function of ß-cells.

Suppression of the human malic enzyme 2 modifies energy metabolism and inhibits cellular respiration.

Human mitochondrial NAD(P)+-dependent malic enzyme (ME2) is well-known for its role in cell metabolism, which may be involved in cancer or epilepsy. We present potent ME2 inhibitors based on cyro-EM structures that target ME2 enzyme activity. Two structures of ME2-inhibitor complexes demonstrate that 5,5'-Methylenedisalicylic acid (MDSA) and embonic acid (EA) bind allosterically to ME2's fumarate-binding site. Mutagenesis studies demonstrate that Asn35 and the Gln64-Tyr562 network are required for both inhibitors' binding. ME2 overexpression increases pyruvate and NADH production while decreasing the cell's NAD+/NADH ratio; however, ME2 knockdown has the opposite effect. MDSA and EA inhibit pyruvate synthesis and thus increase the NAD+/NADH ratio, implying that these two inhibitors interfere with metabolic changes by inhibiting cellular ME2 activity. ME2 silence or inhibiting ME2 activity with MDSA or EA decreases cellular respiration and ATP synthesis. Our findings suggest that ME2 is crucial for mitochondrial pyruvate and energy metabolism, as well as cellular respiration, and that ME2 inhibitors could be useful in the treatment of cancer or other diseases that involve these processes.

Antioxidant Activity and Inhibitory Effects of Black Rice Leaf on the Proliferation of Human Carcinoma Cells.

The leaves of black rice, well-known as postharvest agricultural waste, contain a rich source of antioxidants with multiple benefits for human health. In the present study, the ethyl acetate fraction obtained from black rice leaf was separated into five subfractions using Sephadex LH-20 column chromatography, and their antioxidant and anticancer activities were investigated. The results revealed that among all the subfractions, subfraction 5 (Sub5) showed the highest total phenolic and flavonoid values. The antioxidant activity was also superior in Sub5 (the IC50 values are 3.23, 31.95, and 72.74 µg/mL, in the DPPH, ABTS, and reducing power assays, respectively) compared to the other subfractions. All subfractions, in a time-dependent manner, inhibited the proliferation of hepatoma (HepG2), breast (MCF-7), and colorectal (Caco-2) cancer cells, especially the Sub5. Thus, Sub5 was employed to conduct the cell cycle and cell apoptosis by flow cytometry. Sub5 significantly increased the accumulation of cells at the Sub-G1 phase in HepG2 cells (44.5%, at 48 h). Furthermore, it could trigger annexin V-detected apoptosis through mitochondrial and death receptor pathways accompanied by the suppression of PI3K/Akt and Erk signaling pathways. In addition, HPLC-DAD-MS/MS was conducted to characterize the bioactive constituents in the most potent antioxidant, cytotoxic, and apoptosis-inducing subfraction. Conclusively, Sub5 may have high potential as functional dietary supplements to inhibit the development of HepG2 liver cancer.

Evaluation of Phytochemical Contents and In Vitro Antioxidant, Anti-Inflammatory, and Anticancer Activities of Black Rice Leaf (Oryza sativa L.) Extract and Its Fractions.

Black rice leaves (Oryza sativa L.) are a major part of rice straw left in open fields after rice harvest as agricultural waste. In this study, crude ethanolic extract (CEE) and various solvent fractions (hexane (Hex), ethyl acetate (EtOAc), n-butanol (n-BuOH), and aqueous fractions) of black rice leaves were investigated for their bioactive compound contents as well as antioxidant, anti-inflammatory, and anticancer activities. The results demonstrated that among all the fractions, the n-BuOH fraction presented the greatest contents of total phenolics and flavonoids, while anthocyanins were found to be abundant in the n-BuOH and aqueous fractions, which also exhibited powerful antioxidant abilities according to DPPH and ABTS radical-scavenging assays and a reducing power assay. Regarding anti-inflammatory activity, CEE and EtOAc reduced the production of NO and cytokine secretion (PGE2, IL-6, and IL-1ß) but displayed less effect on tumor necrosis factor α (TNF-α) release in lipopolysaccharide (LPS)-induced RAW 264.7 cells. They also significantly decreased iNOS and COX-2 protein expression. Additionally, the phenolics-rich ethyl acetate fraction showed the greatest activity against HepG2 liver carcinoma cells, inhibited cell growth, increased the Sub-G1 population, and induced apoptosis via mitochondrion-dependent mechanisms. In conclusion, black rice leaves, a byproduct of rice, exhibited strong antioxidant, anti-inflammatory, and anticancer capacities and might be useful for application in functional foods and the pharmaceutical industry.

A Method to Prepare Magnetic Nanosilicate Platelets for Effective Removal of Microcystis aeruginosa and Microcystin-LR.

Algal toxin is a unique type of toxin generated with harmful algal blooms in water bodies. This phenomenon is worsened by eutrophication caused by excessive discharge of nutrients into surface water bodies. Since algal toxins are hard to remove after they enter the water treatment processes, an efficient method is required to inhibit the growth of algal cells, to settle the cells at the bottom of the water body and to removes the toxin from the water. We report an efficient way to prepare a novel nanohybrid material, i.e., magnetic nanosilicate platelet (MNSP), and its effects on the removal of microcystin toxins as well as the cells of Microcystis aeruginosa. MNSP was fabricated by a special treatment of a clay mineral, montmorillonite, and then its surface was decorated with magnetite nanoparticles by in situ synthesis. The nanohybrid can efficiently inhibit the growth of M. aeruginosa-a typical species that can generate one of the most notorious algal toxins, i.e., microcystins. Algal cells can be settled with minimal 500 ppm MNSP, and the turbidity can be reduced by more than 67%. The removal of microcystin-LR (MC-LR) was as high as 99.39% at an concentration of 100 ppm, while the pristine nanosilicate platelet could only remove 36.84% at the same dosage.

Golf ball-assisted electrospray ionization of mass spectrometry for the determination of trace amino acids in complex samples.

During the electrospray ionization (ESI) process, ions move through a heated capillary aperture to be detected on arrival at a mass analyzer. However, the ESI process creates an ion plume, which expands into an ion cloud with an area larger than that of the heated capillary aperture, significantly contributing to an ion loss of 50% due to coulombic repulsion. The use of DC and RF fields to focus ions from the ion source into the vacuum chamber has been proposed in the literature, but the improvement of ion transmission efficiency is limited. To improve ion transmission, in this study we propose a novel method using a home-made golf ball positioned between the ion source and the inlet of the mass analyzer to hydrodynamically focus the ions passing through the golf ball. The ion plume produced by the ESI process passes through the golf ball will reduce the size of the ion cloud then be focused and most of them flowed into the mass analyzer. Therefore, the sensitivity will be improved, the aim of this investigation is to study the enhancing of the signal using golf ball-assisted electrospray ionization liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine 20 trace amino acids in complex samples, including tea, urine and serum. The results showed that the analytical performance of the determination of the 20 amino acids in tea, urine and serum samples using the home-made golf ball-assisted ESI source is better than that of a commercial ESI source. The signal intensities of the 20 amino acids were enhanced by factors of 2-2700, 11-2525, and 31-342680 in oolong tea, urine and serum analyses, respectively. The precision of the proposed method ranged from 1-9%, 0.4-9% and 0.4-8% at low, medium and high concentration levels of amino acids, respectively. The home-made golf ball-assisted ESI source effectively increased the signal intensity and enhanced the ion transmission efficiency and is also an easy, convenient and economical device. This technique can be applied to the analysis of trace compounds in complex matrices.

Down-regulation of heat shock protein 27 in neuronal cells and non-neuronal cells expressing mutant ataxin-3.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. Unstable CAG trinucleotide repeat expansion in the MJD gene has been identified as the pathologic mutation of MJD. In this study, human SK-N-SH neuroblastoma cells stably transfected with full-length MJD with 78 CAG repeats were established. Compared with the parental cells, cells expressing mutant ataxin-3 displayed normal morphology for over 80 generations. Less than 1% of the transfected cells contained nuclear aggregates under basal conditions, indicating that this cellular model represented an early disease stage. While t-butyl hydroperoxide (TBH) was used to assess the oxidative tolerance of cells, the results demonstrated that the transfected cells were more susceptible to low concentrations of TBH than the parental cells. Most interestingly, from 2D gel electrophoresis analysis, we identified that the expression of heat shock protein 27 (HSP27), known as a suppressor of poly(Q)-mediated cell death, dramatically decreased in SK-N-SH cells stably transfected with full-length mutant MJD. The same reduction of HSP27 was further confirmed in lymphoblastoid cells from MJD patients. Our results demonstrated that both neuronal and non-neuronal cells with expanded full-length ataxin-3 revealed reduced protein expression of HSP27. We propose that the reduction of HSP27 in the early stage of the disease plays an important role during cell death process in MJD.

Effective removal of Microcystis aeruginosa and microcystin-LR using nanosilicate platelets.

Drinking water safety has been threatened by increasing harmful algal blooms (HABs) in water sources. HABs are closely associated with eutrophication in freshwater lakes, e.g. Lake Tai in China, and marine environments as well, e.g. Baltic Sea in Europe. Among all HABs, Microcystis aeruginosa attracted much attention due to its easy proliferation and potent toxins, microcystins. Most of the current control technologies can result in immediate release of microcystins which are hard to remove by drinking water treatment processes. Here we propose to simultaneously remove M. aeruginosa and its toxin, microcystin-LR (MC-LR), using nanosilicate platelet (NSP) derived from natural clay mineral. In this study, NSP showed strong selective growth inhibition and good settling enhancing effects on M. aeruginosa and highly efficient removal of MC-LR. NSP can inhibit the growth of M. aeruginosa (initial cell concentration at 3.00×10(6)cellmL(-1)) with a LC50 at 0.28ppm after 12h exposure. At the dosage of 100ppm, NSP can enhance settling of suspended M. aeruginosa. Bacterial growth inhibition tests showed NSP had very mild growth inhibition effects on Escherichia coli at high dosage but promoted the growth of Pseudomonas aeruginosa and Bacillus halodurans. For MC-LR removal, at an initial concentration of 100µgL(-1), NSP achieved higher than 99% removal. Thus, the results suggest that NSP could be an excellent candidate for controlling M. aeruginosa-related HABs in water bodies.

Chemical constituents and anticancer activity of yellow camellias against MDA-MB-231 human breast cancer cells.

Yellow camellia, with its golden yellow flowers, is rare in the world. Most studies of yellow camellia have focused on its ornamental properties; however, there are fewer published studies on its medical values. The purpose of this study was to define the chemical constituents and the biological potential of the water extract of leaves in six species of yellow camellia. The data showed that Camellia murauchii had significantly higher total catechins and total polyphenol content than others; Camellia euphlebia had the highest total amino acids and γ-aminobutyric acid. The results indicated that Camellia tunghinensis exhibited the highest free radical scavenging capacity and showed potent anticancer activities. Camellia nitidissima had stronger inhibitory effect than other species on fatty acid synthesis. In addition to catechins, 3-p-coumaroylquinic acid, kaempferol-3-O-glucoside, and quercetin-3-O-glucoside were detected in C. tunghinensis using liquid chromatography-tandem mass spectrometry. Taken together, yellow camellias possess biological activity and are worthy of continued study.