Radix Rehmanniae Extract Ameliorates Experimental Autoimmune Encephalomyelitis by Suppressing Macrophage-Derived Nitrative Damage

Multiple sclerosis (MS) is a neuroinflammatory disease in central nervous system (CNS) without effective treatment or medication yet. With high prevalence of MS patients worldwide and poor therapeutic outcome, seeking novel therapeutic strategy for MS is timely important. Radix Rehmanniae (RR), a typical Chinese Medicinal herb, has been used for neuroinflammatory diseases in Traditional Chinese Medicine for centuries. However, scientific evidence and underlying mechanisms of RR for MS are unclear. In this study, we tested the hypothesis that RR could attenuate the progress and severity of MS via suppressing macrophage-derived nitrative damage and inflammation by using experimental autoimmune encephalomyelitis (EAE) model for mimicking MS pathology. The results showed the RR treatment effectively ameliorated clinical disease severity, inhibited inflammation/demyelination in spinal cord, and alleviated CNS infiltration of encephalitogenic T cells and activated macrophages. Meanwhile, RR possessed bioactivities of scavenging ONOO− and reducing the expression of iNOS and NADPH oxidases in the spinal cords of the EAE mice. Furthermore, RR treatment suppressed nuclear factor-κB (NF-κB) signaling pathway in the splenocytes of EAE mice. The in vitro experiments on macrophages and neuronal cells exerted consistent results with the in vivo animal experiments. Taken together, we conclude that Radix Rehmanniae extract has therapeutic values for ameliorating EAE/MS pathological process and disease severity and its underlying mechanisms are associated with anti-inflammation and inhibiting macrophage-derived nitrative damages. Further study could yield novel promising therapeutic agent for multiple sclerosis

The molecular mechanism for inhibiting the growth of nasopharyngeal carcinoma cells using polymethoxyflavonoids purified from pericarp of Citrus reticulata ‘Chachi’ via HSCCC

Polymethoxyflavonoids (PMFs), the main bioactive compounds naturally occurring in the pericarp of Citrus reticulata ‘Chachi’ (CRCP), possess significant antitumor action. However, the action of PMFs in nasopharyngeal carcinoma (NPC) is currently unknown. The present research study was conducted to investigate the inhibitory mechanisms of PMFs from CRCP on NPC growth in vivo and in vitro. In our research, we used high-speed counter-current chromatography (HSCCC) to separate four PMFs (nobiletin (NOB), 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), tangeretin (TGN), and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone (5-HPMF)) from CRCP. CCK-8 assay was used to preliminarily screen cell viability following exposure to the four PMFs. Colony formation, Hoechst-33258 staining, transwell, and wound scratch assays were performed to assess the anti-proliferation, invasion, migration, and apoptosis-inducing effects of HMF on NPC cells. NPC tumors in xenograft tumor transplantation experiments were also established to explore the effect of HMF (100 and 150 mg/kg/day) on NPC. The histopathological changes in the treated rats were observed by H&E staining and Ki-67 detection by immunohistochemical techniques. The expressions of P70S6K, p-P70S6K, S6, p-S6, COX-2, p53, and p-p53 were measured by Western blot. The four PMFs were obtained with high purity (>95.0%). The results of the preliminary screening by CCK-8 assay suggested that HMF had the strongest inhibitory effect on NPC cell growth. The results of the colony formation, Hoechst-33258 staining, transwell, and wound scratch assays indicated that HMF had significant anti-proliferation, invasion, migration, and apoptosis-inducing ability in NPC cells. Moreover, HMF suppressed NPC tumor growth in xenograft tumor transplantation experiments. Further investigation suggested that HMF regulated NPC cells proliferation, apoptosis, migration, and invasion by activating AMPK-dependent signaling pathways. In conclusion, HMF-induced AMPK activation inhibited NPC cell growth, invasion, and metastatic potency by downregulating the activation of the mTOR signaling pathway and COX-2 protein levels, as well as enhancing the p53 phosphorylation level. Our study provides a crucial experimental basis for the clinical treatment of NPC, as well as the development and utilization of PMFs from CRCP

Housefly Pupae-Derived Antioxidant Peptides Exerting Neuroprotective Effects on Hydrogen Peroxide-Induced Oxidative Damage in PC12 Cells

In this study, two antioxidant peptides were identified and characterized from the alcalase-hydrolysate of housefly (Musca domestica L.) pupae guided by ABTS cation radical scavenging activity. Peptides sequences were identified as DFTPVCTTELGR (DR12, 1338.48 Da) and ARFEELCSDLFR (AR12, 1485.66 Da) using nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both DR12 and AR12 exert strong ABTS cation radical scavenging ability with EC50 values of 0.39 and 0.35 mM, respectively. Moreover, AR12 can effectively protect PC12 cells from oxidative damage induced by hydrogen peroxide (H2O2) by decreasing intracellular reactive oxygen species (ROS) and malonaldehyde (MDA), recovering cellular mitochondrial membrane potential (MMP), and increasing the activity of intracellular superoxide dismutase (SOD). Stability tests suggest that AR12 is competent for the challenge of heating, acid, alkali or simulated gastrointestinal (GI) digestion and exhibits great activity to remove ABTS cation radical. DR12 shows a great stability against heating, but its antioxidative ability declines after being treated with acid, alkali or simulated GI digestion. In general, both DR12 and AR12 identified from housefly pupae hydrolysate stand a chance of being potential antioxidants or precursors to antioxidants and AR12 might be applied in the field of neuroprotection

Optimization of Extraction of Bioactive Compounds from Baphicacanthus cusia Leaves by Hydrophobic Deep Eutectic Solvents

Deep eutectic solvents (DESs) are considered as efficient and green solvents for the extraction of bioactive compounds from medicinal plants. In this work, a novel method of DES-based ultrasound-assisted extraction of bioactive compounds from Baphicacanthus cusia leaves (BCL) was established. Systematic screening and the morphology of the original and treated BCL were observed with scanning electron microscopy to determine the extraction efficiency of different solvents. The extraction conditions were optimized by Box–Behnken design (BBD) tests and the optimal extraction conditions were as follows: lactic acid/L-menthol ratio of 5: 2 (mol/mol), solid–liquid ratio of 80.0 mL/g and temperature of 60.5 °C. The extraction yields of tryptanthrin, indigo and indirubin reached 0.356, 1.744 and 0.562 mg/g, respectively. The results of a 2,2-diphenyl-1-picrylhydrazy (DPPH) radical scavenging activity test indicated the feasibility of DESs in the extraction of bioactive compounds. This study indicated that L-menthol/lactic acid was a green and efficient solvent for the extraction of bioactive compounds from BCL, and DES-based ultrasound-assisted extraction could be used as an effective application strategy for the extraction of bioactive compounds from medicinal plants

Nobiletin with AIEE Characteristics for Targeting Mitochondria and Real-Time Dynamic Tracking in Zebrafish

Nobiletin is a natural product with multiple physiological activities and is the main ingredient of Pericarpium Citri Reticulatae. We successfully discovered that nobiletin exhibits aggregation induced emission enhancement (AIEE) properties and it has significant advantages such as a large Stokes shift, good stability and excellent biocompatibility. The increase in methoxy groups endows nobiletin a greater fat-solubility, bioavailability and transport rate than the corresponding unmethoxylated flavones. Ulteriorly, cells and zebrafish were used to explore the application of nobiletin in biological imaging. It emits fluorescence in cells and is specifically targeted at mitochondria. Moreover, it has a noteworthy affinity for the digestive system and liver of zebrafish. Due to the unique AIEE phenomenon and stable optical properties of nobiletin, it paves the way for discovering, modifying and synthesizing more molecules with AIEE characteristics. Furthermore, it has a great prospect with regard to imaging cells and cellular substructures, such as mitochondria, which play crucial roles in cell metabolism and death. Indeed, three-dimensional real-time imaging in zebrafish provides a dynamic and visual tool for studying the absorption, distribution, metabolism and excretion of drugs. In this article, more directions and inspiration can be presented for the exploration of non-invasive pharmacokinetic research and intuitive drug pathways or mechanisms

Chemical Fingerprint Analysis and Quantitative Analysis of Saccharides in Morindae Officinalis Radix by HPLC-ELSD

A method based on high performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for the quantitative analysis of three active compounds and chemical fingerprint analyses of saccharides in Morindae officinalis radix. Ten batches of Morindae officinalis radix were collected from different plantations in the Guangdong region of China and used to establish the fingerprint. The samples were separated with a COSMOIL Sugar-D column (4.6 mm × 250 mm, 5 μm) by using gradient elution with water (A) and acetonitrile (B). In addition, Trapped-Ion-Mobility (tims) Time-Of-Flight (tims TOF) was used to identify saccharides of Morindae officinalis radix. Fingerprint chromatogram presented 26 common characteristic peaks in the roots of Morinda officinalis How, and the similarities were more than 0.926. In quantitative analysis, the three compounds showed good regression (r = 0.9995–0.9998) within the test ranges, and the recoveries of the method were in the range of 96.7–101.7%. The contents of sucrose, kestose and nystose in all samples were determined as 1.21–7.92%, 1.02–3.37%, and 2.38–6.55%, respectively. The developed HPLC fingerprint method is reliable and was validated for the quality control and identification of Morindae officinalis radix and can be successfully used to assess the quality of Morindae officinalis radix

Flavanone-Based Fluorophores with Aggregation-Induced Emission Enhancement Characteristics for Mitochondria-Imaging and Zebrafish-Imaging

Fluorophores with aggregation-induced emission enhancement (AIEE) characteristics applied in bioimaging have attracted more and more attention in recent years. In this work, a series of flavanone compounds with AIEE characteristics was developed and applied to fluorescence imaging of mitochondria and zebrafish. The compounds were readily prepared by the thermal dehydration of chalcone that was obtained by the reaction of o-hydroxyacetophenone and benzaldehyde. Two of these compounds showed significant AIEE characteristics by fluorescence performance experiments, including optical spectra, fluorescence spectra, fluorescence quantum yield (φF), fluorescence lifetime, and scanning electron microscopy (SEM). Compared with traditional organic fluorescent dyes, these compounds have high fluorescence emission and high fluorescence quantum yield in solid or aggregated state, which overcomes the shortcoming of aggregation-caused quenching (ACQ). More importantly, the two compounds exhibited low cytotoxicity and good cytocompatibility in A549 lung cells at the experimental concentration range and they specifically targeted mitochondria, which make it of great potential use in mitochondria labeling. In addition, they were embryonic membrane permeable and had different affinities for different tissues and organs of zebrafish, but mainly distributed in the digestive system, providing a basis for the application of such compounds in bioimaging. These AIEE compounds with superior properties could be of great potential use in mitochondria imaging and other in vivo studies

The GC/MS Analysis of Volatile Components Extracted by Different Methods from Exocarpium Citri Grandis

Volatile components from Exocarpium Citri Grandis (ECG) were, respectively, extracted by three methods, that is, steam distillation (SD), headspace solid-phase microextraction (HS-SPME), and solvent extraction (SE). A total of 81 compounds were identified by gas chromatography-mass spectrometry including 77 (SD), 56 (HS-SPME), and 48 (SE) compounds, respectively. Despite of the extraction method, terpenes (39.98~57.81%) were the main volatile components of ECG, mainly germacrene-D, limonene, 2,6,8,10,14-hexadecapentaene, 2,6,11,15-tetramethyl-, (E,E,E)-, and trans-caryophyllene. Comparison was made among the three methods in terms of extraction profile and property. SD relatively gave an entire profile of volatile in ECG by long-time extraction; SE enabled the analysis of low volatility and high molecular weight compounds but lost some volatiles components; HS-SPME generated satisfactory extraction efficiency and gave similar results to those of SD at analytical level when consuming less sample amount, shorter extraction time, and simpler procedure. Although SD and SE were treated as traditionally preparative extractive techniques for volatiles in both small batches and large scale, HS-SPME coupled with GC/MS could be useful and appropriative for the rapid extraction and qualitative analysis of volatile components from medicinal plants at analytical level

Construction and Chemical Profile on “Activity Fingerprint” of Citri Reticulatae Pericarpium from Different Cultivars Based on HPLC-UV, LC/MS-IT-TOF, and Principal Component Analysis

Citri Reticulatae Pericarpium (CRP), known as Chenpi (CP) in Chinese, is a medicinal food for health and fitness. In order to find out the characteristic activity chemicals distinguishing various cultivars of CRP and provide a reference for effective development of citrus resources, an “activity fingerprint” of CRP from 21 different cultivars was established based on the evaluation of antitussive and expectorant activities. There were 18 common peaks in the HPLC fingerprint, of which 3 flavonoid glycosides and 14 polymethoxyflavonoids (PMFs) were identified by LC/MS-IT-TOF. Furthermore, five characteristic chemicals were determined and similarity calculation with principal component analysis (PCA) was combined together to compare the similarity and difference among various cultivars. The results showed that some other cultivars were also similar to C. reticulata “Chachi” except for C. reticulata “Tangerina” and C. reticulata “Dahongpao” recorded in Chinese Pharmacopoeia. Most importantly, the peels of C. reticulata “Shiyueju,” C. reticulata “Ponkan,” C. reticulata “Tribute,” and C. reticulata “Bayueju,” traditionally rarely used for medicinal food, were highly similar to that of C. reticulata “Chachi” and rich in bioactive flavonoids, which can be considered the effective medicinal resources of CRP